13-8400

antibody from Invitrogen Antibodies

Targeting: CTNNB1 armadillo, beta-catenin, CTNNB

Provider product page for 13-8400

Western blot

Immunoprecipitation

Antibody data

Antibody Data
Antigen structure
References [148]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Immunohistochemistry [3]
Flow cytometry [1]
Other assay [64]

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Product number: 13-8400 - Provider product page
Provider: Invitrogen Antibodies
Product name: beta Catenin Monoclonal Antibody (CAT-5H10)
Antibody type: Monoclonal
Antigen: Other
Reactivity: Human, Mouse, Chicken/Avian
Host: Mouse
Isotype: IgG
Antibody clone number: CAT-5H10
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

CTNNB1 protein structure - 13-8400 shown in red.

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Experimental details: Immunohistochemistry analysis of Beta catenin showing staining in the cytoplasm and membrane of paraffin-embedded human colon carcinoma (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Beta catenin monoclonal antibody (Product # 13-8400) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Immunohistochemistry analysis of Beta catenin showing staining in the membrane of paraffin-embedded human stomach tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Beta catenin monoclonal antibody (Product # 13-8400) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Immunohistochemistry analysis of Beta catenin showing staining in the cytoplasm and membrane of paraffin-embedded mouse stomach tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Beta catenin monoclonal antibody (Product # 13-8400) diluted in 3% BSA-PBS at a dilution of 1:100 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Figure 4 Altered Bmp and beta-Catenin Signaling in the K14-Cre; Smo f/f Skin Immunostaining (dark brown) of ventral (A-F and I-L) and dorsal (G, H, and M-P) skin sections from control and mutant mice. (A-F) Phosphorylated Smad 1, 5, and 8 (P-Smad) immunostaining. (B) P-Smad-negative (arrow) and -positive (arrowhead) follicular cells at the skin surface of a mutant. The asterisk in (B) shows a gland. (D-F) Reduced numbers of P-Smad-positive cells in the mutant follicular epithelium (fe). (F) The dermal condensates (dc) associated with the mutant follicles exhibit abnormal nuclear P-Smad staining. (G-O) beta-catenin immunostaining. Nuclear beta-catenin staining in differentiating hair cells (hc) at anagen (G and J) and in the secondary hair germ (hge) at the telogen-to-anagen transition (M) of control follicles. (J) and (K) are high-magnification views of the indicated areas in (I). (H, L, N, and O) Increased beta-catenin staining at cell-cell borders in the interfollicular epidermis (IFE) and in the follicular epithelium (L, N, and O) and decreased staining in the cyst (cy) epithelium. In addition, the mutant follicles are underlain by dc showing nuclear beta-catenin staining. The dashed line in (L) marks the epithelial-mesenchymal junction in a de novo-formed follicle from the IFE. Inset in (L) is a lower magnification view. (P) Shh protein in a newly induced de novo follicle from a pre-existing follicle (arrow). Scale bars: 50 mum (A-D, F-H, J-L, and M-P), 100 mum (E), and 2

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Experimental details: Figure 4 Effect of Hath1, MUC2 or P27Kip1 gene silencing on anchorage independent-growth. A,B, C,D,G,H,I- Filter-grown HT29-Cl.27H, treated or not (control) with DBZ for 21 days, were resuspended and plated on polyHEMA-coated wells (nonadherent conditions) for 72 hours culture in the presence of the indicated siRNA target smart pool (NT, non target; Hath1; MUC2 or P27Kip1). A - RT-PCR detection of Hath1 (left) and Muc-2 (right) mRNA in HT29-Cl.27H cells transfected by siRNA (NT, or Hath1): mean expression relative to siNT DMSO after normalization to actin gene expression; Mean +- SEM of 3 experiments; *, P

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Experimental details: Figure 1 The Wnt pathway is upregulated in drug-resistant cancer cells (A) Western blot analysis of ABCB1, beta-catenin, phosphorylated beta-catenin, GSK3beta, phosphorylated GSK3beta, c-Myc and cyclin D1 protein expression levels in MES-SA, MES-SA/Dx5, MCF-7 and MCF-7/ADR2 cells. (B) beta-catenin transactivation activity assayed by TOP assay in both MDR cells. (C) Quantitative real-time PCR assays of Wnt3A and Wnt5A mRNA expression levels in two MDR cells. (D) Western blot analysis of Wnt5A protein levels in the two MDR cancer cells. Data represent mean +- S.D. of three independent experiments. * P

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Experimental details: Figure 3 Cyclic AMP-dependent protein kinase A pathway is upregulated in two drug-resistant cancer cell lines (A) Real-time RT-PCR analysis of PRKACA, PRKACB and PRKACG mRNA expression in MES-SA, MES-SA/Dx5, MCF7 and MCF7/ADR2 cells. (B) Activities of CRE pathway of 10 muM foskolin-treated MES-SA and MES-SA/Dx5 cells. * P

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Experimental details: Figure 4 CRE and beta-catenin activities and cell viability are reduced in Wnt5A shRNA-knockdown MES-SA/Dx5 cell line (A-B) Western bot analysis of expression levels of (A) Wnt5A, beta-catenin, c-Myc and cyclin D1 and secreted Wnt5A expression levels and (B) beta-catenin, phosphorylated beta-catenin, GSk3,.phosphorylated GSk3beta in shWnt5A transient transfected MES-SA/Dx5 cells. (C) CRE activities and (D) beta-catenin transactivation activities were detected in shWnt5A transient transfected MES-SA and MES-SA/Dx5 cells. (E) Cell cycle assay by PI staining in shWnt5A transient transfected MES-SA/Dx5 cells. (F) Apoptosis rate in doxorubicin-treated Wnt5A-knockdown stably-transfected cells. Data represent mean +- S.D. of three independent experiments. * P

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Experimental details: Figure 7 Antibody depletion of Wnt5A leads to downregulation of PKA/beta-catenin activities and resensitizes doxorubicin toxicity in MES-SA/Dx5 cells (A) Western blot analysis of beta-catenin, phosphorylated beta-catenin, GSK3beta, phosphorylated GSK3beta, c-Myc, and cyclin D1 protein expression levels in Wnt5A antibody-treated MES-SA/Dx5 and MCF7/ADR2 cells. PKA and beta-catenin activities as monitored by (B) CRE and (C) TOP reporter assays, (D) PI staining in cell cycle analysis and (E) Calcein AM assay of drug pump out ability in Wnt5A antibody treated-MES-SA/Dx5 cell. Values are expressed as the mean +- S.D. (n=3). * P

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Experimental details: Figure 6 Tight and adherens junctional proteins in embryonic mouse brain . An overall view of tight and adherens junctional proteins in the ventricular zone of early mouse CSF-brain interface at low magnification in coronal sections of E16 brain showing immunostaining for claudin-5 (A) , alpha-catenin (B) , N-cadherin (C) , and beta -catenin (D) . Note the differences in distribution of immunopositive staining between different regions of the surface of the ventricular zone with strongest staining of the dorsolateral telencephalic wall (DLTW) and a weak or lacking immunoreactivity of the ganglionic eminence (GE). Arrows in A point to the border of faint claudin-5 reactivity of the ventricular surface of the dorsolateral wall, and in (C) to the increased staining of the dorsolateral and ventral borders of the ganglionic eminence. The boxed areas in (A-D) are shown in higher magnification in Figure 7 . HIP, hippocampus; LV, lateral ventricle; SF, septal fork of the lateral ventricle; SVZ, septal ventricular zone. (A-D) Same magnification, scale bar 500 mum.

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Experimental details: Figure 7 Cellular distribution of adherens and tight junctional proteins at the lateral CSF-brain interface . High magnification coronal sections of E16 (A-D) from boxed areas in Figure 6 and of adult (A1-C1) and P20 (D1) forebrain immunostained for claudin-5 (A,A1) , alpha-catenin (B,B1) , N-cadherin (C,C1) , and for beta -catenin (D,D1) . (A) At E16 claudin-5 reactivity is prominent in endothelial cells of blood vessels (BV), but a distinct staining is also present corresponding to the apical-most part of the ventricular zone cells (VZ). (A1) In the adult forebrain ependymal cells (E) show no claudin-5 immunoreactivity in marked contrast to the positively stained endothelial cells of fenestrated blood vessels of the choroid plexus (CP). (B-D) At E16 immunostaining for alpha-catenin (B) , N-cadherin (C) and beta-catenin (D) outlines the apical and apico-lateral most part of the ventricular zone cells,--compare (A) (apical) and (D) (apical and apico-lateral staining). N-cadherin and beta-catenin immunostaining extends into the cytoplasm of the ventricular zone cells ( arrowheads in C,D ). (B1) Immunoreactivity for alpha-catenin is not present in adult forebrain, neither in ependymal cells (E) nor in the choroid plexus (CHP). (C1,D1) The surface of the ependymal cells (E) is strongly stained for N-cadherin but only little reactivity is observed after staining for beta-catenin in the adult forebrain and virtually no reactivity is observed in the subependymal zone. Same magnific

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Experimental details: Figure 1 Analysis of BCL9 expression and canonical Wnt activity in BMECs ( a ) Representative CD34 immunostains in BM biopsies from normal individuals(NBM) (n=20) as well as MGUS (n=20) and MM persons (MMPT) (n=60). Bars: 50mum.( b ) Representative BCL9 immunostains (brown color) in endothelial cells(arrows) in BM biopsies from MM persons (MMPT) or normal bone marrow (NBM) from otherwisehealthy subjects. Selected representative cases are shown. Anti-CD138 staining (red color)is used as a marker of plasma cells on the left panel (arrows). Anti-CD34 staining (redcolor) is used as a marker of endothelial cells (right bottom panel). Bars: 10mum.Immunoblots ( c ) and immunofluorescence ( d ) analysis of BCL9 andbeta-catenin expression in primary endothelial cells derived from BM from two MMpersons (PBMEC 1, PBMEC 1) and two BM endothelial cell lines (BMEC-1, BMEC-60). Noteco-expression of BCL9 (Red color) and beta-catenin (Greed color) by immunoblotting andby nuclear co-localization immunofluorescence. Factor VIII is used as marker ofendothelial cells in immunoblots. Bars: 5mum. ( e ) Wnt reporter activityof BMEC-1, BMEC-60 and PBMEC 1 cells lentivirally transduced with BCL9-shRNA compared withcells lentivirally transduced with scrambled shRNAs (Control-shRNA). ( f )Proliferation of BMEC-1, BMEC-60 and PBMEC 1 cells treated with medium alone (Vehicle) orin the presence of 10 uM SAH-BCL9. Proliferation and Wnt reporter data was normalizedbased on control or vehicle data. Results ar

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Experimental details: FIGURE 6: Uninephrectomy triggered an increase in the size of the remaining kidney. (A) Images of kidneys from 11-wk-old rats. Left, kidney from a control animal; right, kidney from a rat in which the contralateral kidney had been removed 3 wk earlier. (B) Kidney frozen sections from 11-wk-old rats in control condition or that had experimented UNX 1-3 wk earlier. The apical brush border of proximal tubules is stained with specific antibodies against Dpp IV and the basolateral surface with anti-beta-catenin. (C) Quantification of the area of proximal tubule cells from kidneys of 11-wk-old rats (control) or that had experimented UNX 1-3 wk earlier. Area analysis was done using ImageJ on confocal images of frozen kidney sections stained with Dpp IV and anti-beta-catenin. A minimum of 53 cells was evaluated for each condition. Statistical analysis with one-way ANOVA, followed by Dunnett's test; * p < 0.05, *** p < 0.001. (D) ZO-2 expression is lost in the remaining kidneys of rats subjected to UNX. Frozen sections of kidneys from 11-wk-old rats that had been subjected or not to UNX 3 wk earlier were processed for immunofluorescence with an antibody against ZO-2, and the nuclei were stained with DAPI.

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Experimental details: Figure 1 H. pylori CagA and IL-1 beta induce beta -catenin nuclear translocation. (a) MCF-10A cells were infected with CagA positive strains (ABCCC or ABD) or stimulated with IL-1 beta . (b) MCF-10A cells were infected with CagA positive strains and stimulated with IL-1 beta or single infected with CagA negative variants CagA(-) and cagPAI(-). Immunofluorescence images show beta -catenin (green) and nuclei (DAPI, blue). Arrows indicate nuclear staining (a) and membrane staining (b) of beta -catenin. Figures are representative of three independent experiments performed in duplicate or triplicate.

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Experimental details: Figure 3 Nuclear translocation of beta -catenin correlates with increased expression of ZEB1 and Snail1 . (a) Immunoblotting of whole cell lysates of infected or IL-1 beta -stimulated cells with anti- beta -catenin and anti-E-cadherin antibodies. The relative expression of (b) Snail1 and CDH1 and (c) ZEB1 genes was determined by semiquantitative RT-PCR. Expression of housekeeping genes RPLP0 and GAPDH was used as internal control. Figures are representative of three independent experiments performed in duplicate or triplicate. Figures are representative of three independent experiments performed in duplicate or triplicate.

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Experimental details: Figure 2 Wnt/beta-catenin pathway is activated in PIK 3 CA -H1047R tumors. (A) Western blot analysis of mammary tumors from Her2/neu-overexpressing and PIK 3 CA -H1047R transgenic mice. (B) Quantitative RT - PCR of Axin 2 expression in PIK 3 CA -H1047R ( PIK 3 CA - HR )-mutant and Her-2 mammary tumors. The mean (+- SEM ) of mRNA in each tumor in triplicate is shown. Mann-Whitney U -test was performed, and the median of mRNA levels for all tumors is shown on the top right. RPL 19 was used as housekeeping. (C) Western blot and quantification of activated beta-catenin ( ABC ) and beta-catenin proteins in human breast cancer cell line HS 578T overexpressing either vector control (Ctrl) or PIK 3 CA -H1047R and E545K. GAPDH : loading control. (D) Quantitative RT - PCR of the ligand WNT3A (left) and the pathway negative modulator secreted frizzled-related protein 1 ( sFRP -1, right) mRNA in HS 578T control (Ctrl) or mutant PIK 3 CA . Student's t -test was performed, and the mean (+- SEM ) of the triplicate is shown. * P < 0.05; ** P < 0.01; *** P < 0.001.

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Experimental details: Figure 3 Targeting CDH17 reduced the malignant phenotype in gastric carcinoma cells. The expression of CDH17 was suppressed by lentiviral-mediated CDH17 shRNA (shCDH17); cells infected with non-targeted vector (Mock) and parental AGS or MKN-45 cells were used as controls. (A) qRT-PCR(Left) and western blot (Middle) show the mRNA and protein level of CDH17 in different experimental groups after puromycin selection. beta-Actin was used as aloading control. (A) (Right) Growth curves of shCDH17 cells were compared with Mock, AGS cells by XTT assay. (Points, mean of at least three independent experiments; bars, SD). (B) Representative inhibition of foci formation in monolayer culture(Left) and colony formation in soft agar culture(Right) by CDH17 knockdown. Column chart shows quantitative analyses of foci or colonies numbers (columns, mean of at least three independent experiments; bars, SD). (C) (Left) A wound was created on a subconfluent culture of parental AGS, Mock cells and shCDH17 cells, and the rate of wound closure was monitored at 0, 24 and 48 hours. A representative photograph from three independent experiments is shown. Original magnification, x200. (C) (Right) Representative images show the shCDH17, Mock and AGS cells that invaded through the matrigel. The number of invading tumor cells was quantified. Columns, mean of triplicate experiments; bars, SD. Original magnification, x200. (D) Cell cycle analysis by way of flow cytometry. Knockdown CDH17 significantly impaire

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Experimental details: Figure 5 CDH17 activates multiple signal transduction pathways. A. Expression of beta-catenin, GSK-3beta, p- GSK-3beta, Rb, Cyclin D1were compared between shCDH17 and Mock both in AGS and MKN-45 cells by Western blot analysis. MDM2, p53 and p21 also were evaluated. beta-Actin was used as a loading control. B. TOPflash/FOPflash reporter assay shows that Wnt signaling re-activated after CDH17 restoration in AGS and MKN-45. (* P

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Experimental details: Figure 5 Identification of WNT1 and NRP1 as target genes of miR-148a in MDA-MB-231 cells ( A ) Illustration of strategy used to identify target genes of miR-148a. ( B ) qRT-PCR analysis was performed to validate mRNA levels altered by overexpression of miR-148a. ( C ) Protein abundance of WNT1 and NRP1 altered by miR-148a were determined by Western Blot. Two levels of miR-148a overexpression (150x and 20x) were achieved in MDA-MB-231 cells. ( D ) Correlation analysis with linear regression between levels of miR-148a and its target genes was done with GraphPad Prism. The expression levels were extracted from the TCGA database. ( E ) The 3'-UTR of the WNT1 and NRP1 genes contain binding sites for miR-148a seed region according to the TargetScan prediction program.

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Experimental details: Figure 3 Re-morphogenesis of ARPE-19 cells treated with ITS. (A) The photomicrographs show ARPE-19 cells cultured for 4 weeks with ITS and stained for actin with Texas Red-X phalloidin. Note the tight intercellular contact, epithelial-like cellular shape, and predominantly peripheral cortical actin staining. Confocal immunofluorescence images of the same culture conditions showing beta-catenin (B) and N-cadherin (C) at the lateral membrane, CD147 (D) mostly in the apical domain and ezrin (E) in the apical domain. (F) The immunofluorescence image shows immunostaining of the alpha 1 subunit of Na + , K + -ATPase at the lateral membrane and more precisely, as observed in the XZ image, at the cell-cell contacts. (G) The beta 1 subunit is observed at the cell border and at the apical domain; the XZ image confirms both basolateral and apical staining. (H) The beta 2 subunit is observed mainly at the apical domain; the XZ image confirms apical staining. Scale bar: 10 mum. (I) Quantitative analysis of the transepithelial resistance (TER) of ARPE-19 cells during re-morphogenesis is depicted. Note a stable TER of ~80 Omega cm 2 between the second and fourth week in culture.

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Experimental details: Figure 6 Immunocytochemistry of brain endothelial junctional space following growth in either Enriched Media or Minimal Media. Cells were seeded at time 0 h at 20,000 cells per 0.3 cm 2 . ( A ) ECIS resistance and R b measurements over 120 h. Cells were grown in either Enriched Media or Minimal Media from T = 0 h, with media changed at T = 48 h (I). Data shown is the mean +- SD (n = 3 wells) of one independent experiment representative of three experimental repeats; ( B ) Cell number count of Hoechst stained nuclei at time point II, obtained through Image J Software analysis. Data shown is the mean +- SEM (n = 18 wells) of 1 independent experiment representative of 3 experimental repeats; ( C ) The junctional space following growth in either Enriched Media or Minimal Media 72 h post seeding. The time point of fixation corresponds to the second vertical dotted line (II) shown on the ECIS traces. Representative GFP/DAPI and GFP monochrome images for the junctional proteins CD144, ZO-1, beta-catenin, and alpha-catenin are shown. Each panel shows cells grown in Minimal Media on the left and cells grown in Enriched Media on the right. The corresponding Alexa Fluor 488 (GFP) monochrome image is shown below each merged image. Green--junctional protein, blue--nuclei. Scale bar is 200 um. Immunocytochemistry data show one representative image from one independent experiment, which is representative of three experimental repeats. Graphical representations of p values are * p

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Experimental details: Figure 7 Immunocytochemistry of brain endothelial junctional space following growth in either Enriched Media or Minimal Media. Cells were seeded at time 0 h at 20,000 cells per 0.3 cm 2 . ( A ) ECIS resistance and R b measurements over 120 h. Cells were grown in either Enriched Media or Minimal Media from T = 0 h, with media changed at T = 48 h (I). Data shown is the mean +- S.D. (n = 3 wells) of 1 independent experiment representative of 3 experimental repeats; ( B ) Cell number count of Hoechst stained nuclei at time point II, obtained through Image J Software analysis. Data shown is the mean +- SEM (n = 18 wells) of 1 independent experiment representative of 3 experimental repeats; ( C ) The junctional space following growth in either Enriched Media or Minimal Media 72 h post seeding. The time point of fixation corresponds to the second vertical dotted line (II) shown on the ECIS traces. Representative GFP/DAPI and GFP monochrome images for the junctional proteins CD144, ZO-1, beta-catenin and alpha-catenin are shown. Each panel shows cells grown in Minimal Media on the left and cells grown in Enriched Media on the right. The corresponding Alexa Fluor 488 (GFP) monochrome image is shown below each merged image. Green--junctional protein, blue--nuclei. Scale bar is 200 um. Immunocytochemistry data shows 1 representative image from 1 independent experiment, which is representative of 3 experimental repeats. Graphical representations of p values are * p

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Experimental details: Figure 12 ( A ) Western blot representing inhibitory effect of treatments on expression of p-AKT protein in A549 cells. ( B) PFD-D-Lip significantly suppresses p-AKT expression in A549 cells. Cell lysates were prepared following 24-hour treatment of A549 cells with control, PFD and PFD-D-Lip (0.125 mg/mL) and were subjected to western blot analyses using antibodies against p-AKT and actin as a loading control, as indicated. ( C ) Western blot representing inhibitory effect of treatments on expression of beta-catenin protein in A549 cells. ( D ) PFD-D-Lip suppresses beta-catenin expression in A549 cells. Cell lysates were prepared following 72-hour treatment of A549 cells with control, PFD and PFD-D-Lip (0.25 and 0.5 mg/mL) and were subjected to western blot analyses using antibodies against beta-catenin and actin as a loading control, as indicated. The western blot densities were determined and plotted as relative band intensity against treatments. Western blot bands represent representative image of n = 3. Data represent mean +- SD ( n = 3). * p (alpha < 0.05), ** p (alpha < 0.01).

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Experimental details: 3 Figure TRPV4 affects the nuclear localization of beta-catenin and represses beta-catenin/TCF-mediated transcription. (a) Immunofluorescence of endogenous TRPV4 channel (red) and beta-catenin (green) proteins in scattered MDCK cells in control conditions (DMSO) and after activation of the channel either with the addition of 4alpha-PDD (10 uM) for 30 min or by incubation in a hypotonic solution for 45 min. Lower row, cultures preincubated with RN-1734 (30 uM) 10 min before incubation in a hypotonic solution. (xz) Transversal sections at the level indicated with the discontinuous line. Arrows point to cells with nuclear localization of both TRPV4 and beta-catenin. Scale bars = 25 um. (b) MDCK cells were cotransfected with the reporter plasmid (TOPflash or FOPflash), beta-catenin, and increasing amounts (1x, 10x, and 20x) of TRPV4-Flag channel M680K-Flag or Delta235-Flag. Luciferase activity was measured 24 h after transfection. Data correspond to the means +- SEM of the index TOPflash/FOPflash of at least three independent experiments in quadruplicate. (c) Same that in (b) but using the dominant active beta-catenin. For (b) and (c) *** p < .0001; ns, not significant compared with beta-catenin; as determined by one-way ANOVA followed by Dunnett's post hoc test. 4alpha-PDD, 4alpha-phorbol 12, 13-didecanoate; ANOVA, analysis of variance; DMSO, dimethyl sulfoxide; MDCK, Madin-Darby canine kidney; SEM , standard error of the mean; TRPV4, transient receptor potential vanilloid 4

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Experimental details: 4 Figure The interaction between TRPV4 and beta-catenin is modulated by the channel activity. (a) Coimmunoprecipitation of TRPV4-Flag and Myc-beta-catenin in transiently transfected MDCK cells. Interaction of beta-catenin TRPV4-Flag (up), M608K-Flag (middle), and Delta235-Flag (down). Proteins immunoprecipitated with anti-Flag (Flag IP) or with anti-Myc (Myc IP) antibodies were analyzed by western blot to detect TRPV4 (anti-Flag) and beta-catenin (anti-Myc). IgG, unrelated Ab as negative control; Input, 1% of the protein lysate. Immunoprecipitated proteins are shown in a separate line on the right, although they were analyzed in the same gel with IgG and input controls. For M680K and Delta235 Flag IP lines, a short exposure of the immunoprecipitated channel is compared with a long exposure of the IgG and input lines from the same membrane. (b) Interaction between endogenous TRPV4 and beta-catenin in MDCK cells after one (1d), three (3d), or seven days (7d) in culture after being seeded at 2 x 10 4 cells/cm 2 . PLA signal was detected as red dots by confocal microscopy. Immunodetection of the tight junction marker protein ZO-1 (green) is shown to visualize the cell-cell borders. Scale bars = 25 um. (c) Immunofluorescence of endogenous TRPV4 channel (red) and beta-catenin (green) in MDCK cells after 7 days of culture. Nuclei were stained with Hoechst. Scale bar = 25 um. (d) PLA signal of TRPV4 and beta-catenin interaction in scattered MDCK cells after activation of TRPV4. MDCK

Supportive validation

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Experimental details: 5 Figure TRPV4 contains more than one beta-catenin interaction domain. (a) Diagram showing the N-terminus of TRPV4 (1-465 aa) with the six ankyrin domains as well as the Pacsin 3 and OS-9 interaction domains. The lower part shows different TRPV4 constructs tested for interaction with beta-catenin and the location of the Flag epitope. (b) MDCK cells were cotransfected with the reporter plasmid TOPflash or FOPflash, beta-catenin, and increasing molar amounts of 1-235, 1-410, or Delta410-Flag. Luciferase activity was evaluated 24 h after transfection. Data correspond to the means +- SEM of the index TOPflash/FOPflash of at least three independent experiments in quadruplicate. * p < .05; *** p < .0001 compared with beta-catenin. One-way ANOVA and Dunnett's post hoc test. (c) Coimmunoprecipitation of the peptides of TRPV4 and Myc-beta-catenin transiently transfected in MDCK cells. Interaction of beta-catenin with 1-235, 1-410, and Delta410. Proteins immunoprecipitated with anti-Flag antibody (IP: Flag) were analyzed by western blot to detect beta-catenin (anti-Myc) and TRPV4 peptides (anti-Flag). Cotransfection of the empty vector was used as a negative control. Input, 15 ug of total protein lysate. ANOVA, analysis of variance; MDCK, Madin-Darby canine kidney; SEM , standard error of the mean; TRPV4, transient receptor potential vanilloid 4

Supportive validation

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Experimental details: Fig. 6 miR-485-5p targets CLDN4 to regulate the Wnt/beta-catenin signaling. A, binding site between miR-485-5p and CLDN4 predicted on Starbase ( http://starbase.sysu.edu.cn/ ); B, miR-485-5p expression profile in OC predicted on GEPIA ( http://gepia.cancer-pku.cn/ ); C, protein expression of CLDN4 in tumor tissues and the paired normal tissues determined by immunohistochemical staining (paired t test,** p < 0.01); D, CLDN4 expression in OC cells and SV40 cells measured by RT-qPCR; E-F, binding relationship between miR-485-5p and CLDN4 validated through a dual luciferase reporter gene assay (E) and an RIP assay (F) (two-way ANOVA,* p < 0.05); G, CLDN4 expression in OC cells after miR-485-5p mimic and LV-CLDN4 transfection determined by RT-qPCR (one-way ANOVA, * p < 0.05 compared to NC mimic, # p < 0.05 compared to miR-485-5p mimic + LV-NC); H, protein levels of Wnt1 and beta-catenin in OC cell lines after miR-485-5p mimic and LV-CLDN4 transfection evaluated using western blot analysis (two-way ANOVA,* p < 0.05 compared to NC mimic, # p < 0.05 compared to miR-485-5p mimic + LV-NC). Data were exhibited as mean +- SD from three independent experiments.

Supportive validation

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Experimental details: Figure 1 Sorbs2 (sorbin and SH3 domain-containing 2) is a cardiac-enriched intercalated disc and desmosome protein in mice. A , Western blot analysis of Sorbs2 protein expression in different mouse tissues. B , Images of immunostaining using an anti-Sorbs2 antibody and counterstained with 4',6-diamidino-2-phenylindole (DAPI) in sectioned wild-type mouse hearts. Arrowheads indicate the intercalated disc. The arrows indicate Z-disc localization. Red arrows indicate the perinucleus. Bar=20 mum. C , Immunostaining and pseudoline analysis of sectioned mouse hearts are shown, indicating that Sorbs2 protein mostly colocalizes with beta-catenin and plakoglobin (Pkg), but less with connexin 43 (Cx43). Bar=2 mum. MW indicates molecular weight.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: 4 FIGURE DEGs between male and female cases in NAFLD using Wnt focusing RT 2 profiler Polymerase chain reaction (PCR) arrays. (a) Scatter plot comparing the normalized average expression of each gene of the ""Wnt targets"" PCR array between the two groups (cases 8 & 5 ""NAFLD"" vs. cases 2 & 3 ""normal"" in female samples). The red and green dot stand for up-regulated and down-regulated genes, respectively, whereas black dots indicate unchanged genes; cut-off = +-1.5. (b) Visualization of log2 (Fold Change). Two groups of differential genes in female samples, intersect and merge gene expression colorimetry maps. Based on the expression levels of the DEGs in each in groups, a log2 is taken. Using systematic clustering (Hierarchical Cluster), the overall clustering result of the sample is obtained. Red indicates higher expression and blue indicates lower expression. (c) Scatter plot comparing the normalized average expression of each gene of the ""Wnt targets"" PCR array between the two groups (cases 6 & 7 ""NAFLD"" vs. cases 1 & 4 ""normal"") in male samples. (d) Visualization of log2 (Fold Change) of gene expression in male samples. (e) Protein expression of beta-catenin, FRZB, PITX2 and WIF1 in normal and NASH samples analyzed by immunoblotting. The samples include Normal 1 (male), Normal 2 (female), NASH 1 (male) and NASH 2 (female). Primary antibodies against beta-catenin, FRZB, PITX2 and WIF1 were used for detection. beta-Actin was used as the loading (reference) control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 miR-26a-5p overexpression modulated expression of Wnt5A/beta-catenin signaling mediators and downstream molecules in UTUC cells. BFTC-909 cells were transfected with miR-26a-5p mimics for 48 h and subjected to detection of mRNA and protein expression by using qPCR ( A ) and western blot ( B) , respectively. GAPDH was used as loading internal control. ( C ) The blotting densitometry of protein levels in BFTC-909 cells. Data are expressed as mean +- SEM (n = 3). *Indicates a P < 0.05 between the indicated groups.

Supportive validation

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Experimental details: Changes in expression levels of FYN and BTB/apical ES-associated proteins in mouse testes during postnatal development. Male mice at 2-10 weeks old were terminated to obtain lysates of testes for immunoblotting experiments (A) . The steady-state levels of TJ proteins (occludin and CAR), ES proteins (N-cadherin, beta-catenin and nectin-3), SRC family kinases (including FYN and another two family members SRC and YES, which are known to regulate BTB and apical ES), and cytoskeletal proteins (including actin and its binding proteins Arp3, Eps8, and annexin A2, as well as alpha- and beta-tubulin) were examined. GAPDH was used as an internal control to ensure the equal protein loading across the gel (~50 ug protein/lane). Histograms (B) summarizing the results of immunoblot analysis in (A) . Each data point was first normalized against its corresponding GAPDH result, then against that of "postnatal week 2" (except for nectin-3, for which "postnatal week 3" was used), which was arbitrarily set to 1. Each bar = mean +- S.D. of three independent experiments. *P

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 FYN is an integral component of the BTB and apical ES as demonstrated by Co-IP and dual-labeled IF assays. (A) Co-IP assays to determine the physical interactions between FYN and selected BTB/apical ES-associated proteins [including TJ proteins CAR (A, a) and occluding (A, b) , basal ES protein beta-catenin, apical ES protein nectin-3, as well as cytoskeletal proteins actin, alpha-tubulin, Arp3, Eps8, and annexin A2] using mouse testis (T) lysates (~800 ug total protein/sample). A positive control (+ve Ctrl) was either testis lysate (30-50 ug protein/lane) without Co-IP, or the immunoprecipitated FYN was detected using its own antibody by immunoblotting (A, a) . For the negative control (-ve Ctrl), anti-FYN IgG was replaced with rabbit (Rbt) IgG. Several immunoblots show the heavy (IgG H ) and light (IgG L ) chains of IgG to better illustrate Co-IP results. (B) Dual-labeled IF staining to corroborate FYN localization at the BTB by its co-distribution with TJ proteins occludin and ZO-1, as well as basal ES protein beta-Catenin in the testis. Scale bar, 10 mum. (C) FYN co-localization with actin binding protein Arp3, a key regulator of branched actin nucleation at the BTB and apical ES, further substantiated its presence at the BTB and apical ES (). The stage of the seminiferous epithelium is indicated in Roman numerals. Scale bar, 16 mum. Boxed areas denoted with i and ii (showing FYN-Arp3 co-localization at the BTB and apical ES, respectively) are enlarged and presen