PA1-183

antibody from Invitrogen Antibodies

Targeting: ACTB

Provider product page for PA1-183

Western blot

Immunocytochemistry

Immunoprecipitation

Other assay

Antibody data

Antibody Data
Antigen structure
References [46]
Comments [0]
Validations
Western blot [2]
Immunocytochemistry [1]
Other assay [35]

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Product number: PA1-183 - Provider product page
Provider: Invitrogen Antibodies
Product name: beta Actin Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Purifed from natural sources
Description: By Western blot, PA1-183 detects a prominent ~42kD protein in human, mouse, non-human primate, and rat whole cell lysates. For visualization of actin filaments by immunofluorescence, fixation and permeabilization with methanol is required.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 100 µg
Concentration: 1 mg/mL
Storage: -20° C, Avoid Freeze/Thaw Cycles

ACTB protein structure - PA1-183 shown in red.

Supportive validation

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Experimental details: Western blot analysis of beta-actin was performed by loading 25 µg of various whole cell lysates and 10 µL of PageRuler Plus Prestained Protein Ladder (Product # 26619) per well onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. The membrane was probed with a beta-actin polyclonal antibody (Product # PA1-183) at a dilution of 1:1000 overnight at 4°C on a rocking platform, washed in TBS-0.1%Tween-20, and probed with a goat anti-rabbit IgG-HRP secondary antibody (Product # 31460) at a dilution of 1:40,000 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

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Experimental details: Western Blot was performed using Anti-beta Actin Polyclonal Antibody (Product # PA1-183) and a ~40 kDa band corresponding to Actin, cytoplasmic 1 was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A549 (Lane 2), A-431 (Lane 3), U-2 OS (Lane 4), Jurkat (Lane 5), PC-12 (Lane 6), NIH/3T3 (Lane 7), Mouse Spleen (Lane 8), Mouse Liver (Lane 9), Rat Spleen (Lane 10), Rat Liver (Lane 11) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2001) by iBlot® 2 Dry Blotting System (Product # IB21001). The Blot was probed with the primary antibody (1:2000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). doi:10.1038/bcj.2014.18

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Experimental details: Immunoprecipitation of beta-actin was performed on HeLa cells. Antigen-antibody complexes were formed by incubating 750 µg of HeLa whole cell lysates with 2 µg of a beta-actin polyclonal antibody (Product # PA1-183) overnight on a rocking platform at 4øC. The immune complexes were captured on 50 µL Protein A/G Agarose (Product # 20421), washed extensively, and eluted with 5X Lane Marker Reducing Sample Buffer (Product # 39000). Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween-20 for at least 1 hour. The membrane was probed with a beta-actin polyclonal antibody (Product # PA1-183) at a dilution of 1:1000 overnight rotating at 4øC, washed in TBST, and probed with Clean-blot IP detection reagent (Product # 21230) at a dilution of 1:250 for at least 1 hour. Chemiluminescent detection was performed using SuperSignal West Pico (Product # 34080).

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Supportive validation

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Supportive validation

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Experimental details: Figure 3 Activation of p38 MAPK pathway on A549 after betaine and/or phycocyanin treatment. Cells were treated with either 4% betaine, 20 mu g*L -1 of C-PC, or a combination of both molecules for 24 and 48 h. (a) Expression of p38 MAPK in different treatments was monitored by western blot. beta -actin was used as a loading-control protein. (b) p38 MAPK along with beta -actin band signals were quantified with the ImageJ software. After normalization with beta -actin, p38 MAPK values were plotted on the graph as relative values compared to their respective controls. Bars are presented as the mean +- standard deviation. * p < 0.05. Statistical significance between groups was assessed using repeated measures one-way ANOVA followed by Tukey's posttest. UT stands for untreated condition, B stands for betaine, and PC stands for C-PC.

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Experimental details: 2 FIGURE Assessment of catabolic activity in disc tissue of 23-month-old PAPPA -/- mice compared to 23-month-old Wt mice. Western blotting and densitometry measurements were performed as described in Section 2. A, A schematic of the mouse aggrecan core protein covalently linked to the sulphate-rich GAG and noncovalently bound to a hyaluronan chain via the link protein. The cleavage sites between G1 and G2 interglobular domains by ADAMTS (G1-NVTEGE392) and MMP (G1VDIPEN360) proteases are indicated. B, Western blot of 23-month-old PAPPA -/- and 23-month-old Wt disc tissues probed for aggrecan fragments. Six-month-old Wt tissues serve as young controls. Arrows indicate two specific cleavage products corresponding to ADAMTS (~65 kD) and MMP (~55 kD). Three replicates per condition. C, Quantification of aggrecan fragment western blot data for 6-month-old Wt, 23-month-old Wt, and 23-month-old PAPPA -/- tissues. Bars represent mean values of three different mouse tissues; error bars indicate SD. All values were normalized to beta-actin control. *, P < .05. Abbreviations: disc, intervertebral disc; MMP, matrix metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; Wt, wild-type

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Experimental details: 3 FIGURE Assessment of catabolic protease expression in disc tissue of 23-month-old PAPPA -/- mice compared to 23-month-old Wt mice. Data shown are representative immunoblots and densitometric quantification of MMP-3 (A) and ADAMTS-4 (B) protein expression. Bars represent mean values of three different mouse tissues; error bars indicate SD, n = 3. All values were normalized to beta-actin control. *, P < .05

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Experimental details: 4 FIGURE Assessment of disc cellular senescence in disc tissue of 23-month-old PAPPA -/- mice compared to 23-month-old Wt mice. Immunohistochemistry slides were prepared as described in Section 2. Representative examples are shown. A, Lamin B1 staining for qualitative assessment of senescent disc cells. There was higher intensity of lamin B1 staining in PAPPA -/- mice as indicated by the black arrows. No detectable lamin B1 staining was seen in 23-month-old Wt mice samples. B, Western blotting probed for p53 protein expression. Three replicates per condition. C, Densitometric quantification of p53 immunoblots of PAPPA -/- mice showed a decrease in p53 expression, albeit not statistically significant ( P = .12), compared to 23-month-old Wt mice. Bars represent mean values of three different mouse tissues; error bars indicate SD. All values were normalized to beta-actin control

Supportive validation

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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: Figure 6 EPEC infection model increases intestinal permeability and decreases claudin-1 expression in colon of mice. (A) Intestinal permeability was determined in the plasma of control and EPEC-infected mice at day 7 p.i. using FITC dextran assay. Each point represents a mouse. Lines represent mean +- SEM (n=8). * p < 0.006 using Student's t-test . (B) Significant positive correlation between diarrhea score and INF-gamma levels in colon tissues of EPEC-infected mice at day 7 p.i (Spearman rank test). (C) . Representative western blots of levels of claudin-1 and beta-actin in colonic tissues of control and EPEC-infected mice at day 7 p.i. (D) Quantification of western blot bands of claudin-1 and beta-actin. Bars represent mean +- SEM (n=3). # p < 0.03 using Student's t-test . (E) Representative western blots of levels of claudin-2 and beta-actin in colonic tissues of control and EPEC-infected mice at day 7 p.i. (F) Quantification of western blot bands of Claudin-2 and beta-actin. Bars represent mean +- SEM (n=3).

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Experimental details: Figure 3. Bcl2l10-knockdown significantly decreases the protein expression levels of SDHD and IDH1. (A) SKOV3 and A2780 cells were transfected with siNC or siBcl2l10 and harvested 48 h after transfection. The protein expression levels of BCL2L10, SDHD, IDH1 and FH were measured by western blot analysis using beta-actin as an internal control gene. (B) Protein signals of BCL2L10, SDHD, IDH1 and FH were measured and normalized to the corresponding beta-actin signals. The relative expression levels of proteins were calculated by comparing all the normalized signals to that of the negative control group. All the experiments were repeated at least three times, and the data are expressed as the mean +- SEM. *P

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Experimental details: Figure 4. Bcl2l10-knockdown significantly increases TPM4 expression. (A) SKOV3 and A2780 cells were transfected with siNC or siBcl2l10 and harvested 48 h following transfection. mRNA expression levels of TPM4 were calculated from CT values obtained by reverse transcription-quantitative PCR and were normalized to the expression levels of GAPDH. (B) Protein levels of TPM4 was measured by western blot analysis. beta-actin was used as an internal control gene. Signals of TPM4 were measured and normalized to corresponding beta-actin signals. Relative expression levels of TPM4 were calculated by comparing all the normalized signals to that of the negative control group. All experiments were repeated at least 3 times, and data are expressed as the mean +- SEM. *P

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Experimental details: Figure 1 SIRT1 was down-regulated in the STZ-induced DR rat model. STZ-treated rats were further treated with DMSO, 10 mg/kg CMS, 50 mg/kg CMS, and 100 mg/kg CMS. n = 15 per treatment. ( A ) Representative images of retinal tissues observed by HE-staining (x 400) (scale bar = 25 mum) and quantitative analysis of thickness. * p < 0.05 compared to sham-operated rats and # p < 0.05 compared to STZ-treated rats. ( B ) Ultrastructure of rat retinal tissues observed under a TEM (x 10000) (scale bar = 1 mum). The arrow represents the capillary BMT, which is used to measure basement membrane width. ( C ) Expression pattern of SIRT1 measured by RT-qPCR in rat retinal tissues, normalized to beta-actin. ( D ) Representative Western blots of SIRT1 protein and its quantitation in rat retinal tissues, normalized to beta-actin. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the sham-operated rats, and # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the rats injected with STZ and treated with DMSO. The results were measurement data and expressed as mean +- standard deviation. Comparisons between multiple groups were analyzed by one-way ANOVA with Tukey''s post hoc test. (n = 15). DR, diabetic retinopathy; STZ, streptozotocin; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SIRT1, Sirtuin 1; TEM, transmission electron microscopy; ANOVA, analysis of variance; n, number; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nu

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Experimental details: Figure 3 CMS reduced oxidative stress, inflammation, and apoptosis in DR rats. STZ-treated rats were treated with DMSO, 10 mg/kg CMS, 50 mg/kg CMS, and 100 mg/kg CMS. n = 15 per treatment. ( A ) Expression pattern of iNOS as determined by RT-qPCR in rat retinal tissues, normalized to beta-actin. ( B ) Representative Western blots of iNOS protein and its quantitation in rat retinal tissues, normalized to beta-actin. ( C ) Expression of NO measured by nitrite test in rat retinal tissues. ( D - F ) Expression patterns of IL-6 ( D ), TNF-alpha ( E ), and CRP ( F ) measured by ELISA in the cell supernatant. ( G , H ) Representative images of apoptotic cells (x 400) (scale bar = 25 mum) ( G ) and cell apoptosis in rat retinal tissues ( H ) by TUNEL. ( I ) Representative Western blots of cleaved caspase-3 protein and its quantitation in rat retinal tissues, normalized to beta-actin. ( J , K ) Representative Western blots of Cyt-C protein and its quantitation in the cytoplasm and mitochondria, normalized to beta-actin. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the sham-operated rats, and # p < 0.05, ## p < 0.01, ### p < 0.001, compared to the rats injected with STZ and treated with DMSO. The results were measurement data, which were expressed as mean +- standard deviation. Comparisons between multiple groups were analyzed by one-way ANOVA with Tukey''s post hoc test (n = 15). iNOS, inducible nitric oxide synthase; CMS, coumestrol, STZ, streptozotocin; DMSO, dimethyl sulfoxi

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Experimental details: Figure 4 CMS activated the expression of SIRT1 in HG-treated hRMECs. hRMECs were transfected with sh-SIRT1-1, sh-SIRT1-2, or sh-SIRT1-3, and HG-exposed hRMECs were treated with DMSO, 1-CMS, 2-CMS, or 3-CMS, respectively. ( A ) Cell viability assessed by CCK-8 assay. ( B ) SIRT1 expression pattern determined by RT-qPCR in hRMECs, normalized to beta-actin. ( C ) Representative Western blots of SIRT1 protein and its quantitation in hRMECs, normalized to beta-actin. ( D , E ) Representative images (x 400) (scale bar = 25 mum) ( D ) as well as SIRT1 activity and nuclear accumulation in hRMECs ( E ) detected by immunofluorescence staining. ( F ) Expression pattern of SIRT1 as determined by RT-qPCR in hRMECs, normalized to beta-actin. ( G ) Representative Western blots of VEGF protein and its quantitation in hRMECs, normalized to beta-actin. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to the control cells, and # p < 0.05, ## p < 0.01, ### p < 0.001, compared to cells stimulated with NC or HG + DMSO. The results were measurement data and expressed as mean +- standard deviation. Comparisons between multiple groups were analyzed by one-way ANOVA with Tukey''s post hoc test. The cell experiments were repeated three times independently. NC, negative control; CMS, coumestrol, HG, high glucose; DMSO, dimethyl sulfoxide; SIRT1, sirtuin 1; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; VEGF, vascular endothelial growth factor; ANOVA, analysis of variance.

Supportive validation

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Experimental details: Figure 6 CMS suppressed HG-induced oxidative stress and inflammation in hRMECs through activating SIRT1. hRMECs were treated with sh-NC, sh-SIRT1, sh-SIRT1 + DMSO and sh-SIRT1 + CMS, respectively. ( A ) Expression pattern of iNOS as determined by RT-qPCR in hRMECs, normalized to beta-actin. ( B ) Representative Western blots of iNOS protein and its quantitation in hRMECs, normalized to beta-actin. ( C ), Expression pattern of NO as determined by nitrite test in hRMECs. ( D - F ), Expression patterns of IL-6 ( D ), TNF-alpha ( E ), and CRP ( F ) as measured by ELISA in the cell supernatant. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to sh-NC-treated cells, and # p < 0.05, ## p < 0.01, ### p < 0.001, compared to cells stimulated with sh-SIRT1 and treated with DMSO. The results were measurement data and expressed as mean +- standard deviation. Comparisons between multiple groups were analyzed by one-way ANOVA with Tukey''s post hoc test. The cell experiments were repeated three times independently. NC, negative control; CMS, coumestrol, HG, high glucose; hRMECs, human retinal microvascular endothelial cells; SIRT1, sirtuin 1; DMSO, dimethyl sulfoxide; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; IL-6, interleukin-6; TNF-alpha, tumor necrosis factor alpha; CRP, C-reactive protein; ELISA, Enzyme linked immunosorbent assay; ANOVA, analysis of variance.

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Experimental details: Figure 7 CMS suppressed the apoptosis of HG-treated hRMECs . hRMECs were treated with sh-NC, sh-SIRT1, sh-SIRT1 + DMSO and sh-SIRT1 + CMS, respectively. ( A ) Cell cycle distribution as determined by flow cytometry analysis. ( B ) Cell apoptosis as determined by flow cytometry analysis. ( C ) Representative Western blots of cleaved caspase-3 protein and its quantitation in hRMECs, normalized to beta-actin. ( D , E ) Western blots of Cyt-C protein ( D ) and its quantitation ( E ) in the cytoplasm and mitochondria, normalized to beta-actin. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to sh-NC-treated cells, and # p < 0.05, ## p < 0.01, ### p < 0.001, compared to cells stimulated with sh-SIRT1 + DMSO. The results were the measurement data and expressed as mean +- standard deviation. Comparisons between multiple groups should be analyzed by one-way ANOVA with Tukey''s p ost hoc test. The cell experiments were repeated three times independently. CMS, coumestrol; HG, high glucose; hRMECs, human retinal microvascular endothelial cells; NC, negative control; DMSO, dimethyl sulfoxide; SIRT1, sirtuin 1; NO, nitric oxide; Cyt-C, cytochrome c; ANOVA, analysis of variance.

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Experimental details: Figure 3 ( A ) Brain lysates from WY, WO, GY, and GO mice were collected, and levels of SMP30 were analyzed by Western blotting. ( B ) Beta-actin was used to normalize Western blot data ( n = 8). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT -/- mice (GY); old GNMT -/- mice (GO).

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Experimental details: Figure 5 ( A ) Brain lysates from WY, WO, GY, and GO mice were collected, and levels of ADNP were analyzed by Western blotting. ( B ) Beta-actin was used to normalize Western blot data ( n = 8, *** p < 0.001). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT -/- mice (GY); old GNMT -/- mice (GO).

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Experimental details: Figure 7 ( A ) Brain lysates from Wt, GY, and GO mice were collected, and levels of RIPK1 were analyzed by Western blotting. The RIPK1 expression was down-regulated or non-detectable in some WY and WO mice; thus, the WY and WO mice were combined into one group as Wt. ( B ) Beta-actin was used to normalize Western blot data ( n = 8, * p < 0.05 and ** p < 0.005). Abbreviations: wild-type mice (Wt); young GNMT -/- mice (GY); old GNMT -/- mice (GO).

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Experimental details: Figure 9 ( A ) Brain lysates from WY, WO, GY, and GO mice were collected, and levels of Caspase 3 were analyzed by Western blotting. ( B ) Beta-actin was used to normalize Western blot data ( n = 8). Abbreviations: young wild-type mice (WY); old wild-type mice (WO); young GNMT -/- mice (GY); old GNMT -/- mice (GO).

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Experimental details: Figure 4 Effect of KDAC inhibition on enzyme activities in A549 cells under normoxia and hypoxia. ( A , B ) The ATP- dependent 6-phosphofructokinase (PFK1) ( A ) and lactate dehydrogenase (LDH) ( B ) enzymatic activities were measured after 24 h of incubation, and activities were normalized to intracellular protein content in each condition. A549 cells were treated with 1 muM of TSA, 20 mM of NAM, and both 1 muM TSA and 20 mM NAM for 24 h of incubation under normoxia and hypoxia. Cells incubated in medium without KDACIs served as control. Bars represent the means +- standard error of the mean of three independent experiments. The asterisks above bars indicate statistically significant differences compared to normoxic control cells. The asterisks above curly brackets indicate statistically significant differences between hypoxic and normoxic treatments and between hypoxic treatments and hypoxic control cells. Statistical significance was assessed by a two-tailed Student''s t -test. *, p

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Experimental details: Figure 1 Degenerative changes in intervertebral discs of naturally aged mice. (a) Expression of selected senescent markers, p16 Ink4a (a1), p53 (a2) and p21 (a3) in disc tissue from young (6 month) and old (22 month) mice was determined by Western blotting and qRT-PCR. Graphs on the right of the Western images are quantification of results (a2 and a3) whereby the volume of respective protein band divided by volume of beta-actin band. (b) Increased disc aggrecan proteolysis with age. (b1) A schematic of the mouse aggrecan core protein covalently linked to the sulphate-rich glycosaminoglycan (GAG) and noncovalently bound to a hyaluronic acid (HA) chain via the link protein. The cleavage sites between G1 and G2 interglobular domains by ADAMTS (G1-NVTEGE 392 ) and MMP (G1VDIPEN 360 ) proteases are indicated. (b2) Western blot analysis of aggrecan fragments generated by ADAMTS and MMP proteases with graphs showing quantification results. Representative Western blots for three young and three old mice were shown (panels a2, a3, b2). (c) Proteoglycan synthesis as measured by 35 S-sulphate incorporation using whole disc organ cultures. Student's t test was used for parametric data (graphs a1, a2, b2), and the Mann-Whitney test was used for two-variable nonparametric data (graphs a3, c). Data are means +- SD of four independent experiments (4 mice) for graphs a1, a3, c and three mice for graphs a2, b2. * p < 0.05. Y = young and O = old. Representative Western images of three different

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Experimental details: Fig. 4 Effects of DAPK3 inhibition on CLL and lymphoid cell proliferation and cellular function. (A) Representative western blots displaying levels of EGR1 and DAPK3-T265-P in CLL cells pretreated with 0-120 mu m DAPKi for 1 h and with or without anti-IgM stimulation for 1 h. beta-actin was used as a loading control. Blots are normalised to unstimulated sample (unstimulated = 1.0) with image lab software (below). Error bars represent the SD of three independent experiments. Significant differences calculated using two-way ANOVA followed by Dunnett's multiple comparison test with anti-IgM stimulated 0 u m sample as control. P values = 0.0076, 0.0256 and 0.0087 for unstimulated 0 and 80 and 120 u m samples, respectively. (B) Flow cytometric analysis of CLL cells cultured on a CD40L-expressing cell feeder layer. CLL cells were pretreated with either 1 u m ibrutinib (green), increasing concentrations (5-80 u m ) of DAPKi (red), both inhibitors (green/red), or DMSO (white; negative control) for 1 h as indicated. Proliferation was assessed via the percentage of cells positive for Ki-67. Data shown are representative of n = 3 CLL patients. (C) Flow cytometric analysis of CLL cells cultured on a CD40L-expressing cell feeder layer. CLL cells were pretreated with either 1 u m ibrutinib, increasing concentrations (5-80 u m ) of DAPKi or DMSO (negative control) for 1 h. Apoptosis was assessed using the Annexin V Apoptosis Detection Kit, as indicated. Proliferation was assessed via the pe

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Experimental details: Figure 3 Mechanistic evaluation of SDC1-Lip echinomycin-induced autophagic (non-apoptotic) cell death using Western blot. As observed in Figure 2 A,B, SDC1-Lip echinomycin induced cell death. To evaluate potential mechanisms of cell death, S2VP10, S2CP9, and Miapaca2 cells were treated with PBS, Gemzar, echinomycin alone, or SDC1-Lip echinomycin. Cell lysates were evaluated for signaling molecules important in autophagy (LC3-I, -II, p62, ATG7, and ATG12) and apoptosis (pro-caspase-3, cleaved caspase-3, pro-caspase-9, and cleaved caspase-9). ( A - F ) Echinomycin alone and ( G - L ) SDC1-Lip echinomycin treatment of S2VP10 and S2CP9 showed increases in the LC3-II/I ratio, indicating macroautophagy and lack of cleaved caspases-3 and -9, thus no apoptosis. Miapaca2 cells did not demonstrate activation of autophagy in response to either echinomycin or SDC1-Lip echinomycin. Control apoptosis treatment, Gemzar, resulted in some activation of apoptosis. Quantification comparisons are shown in B-F and H-L as well as Figure S6A-D . * p < 0.05.

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Experimental details: Figure 4 miR-375 regulates ERBB2 and VEGFA in EC . A. Expression of miR-375 after transfection. B. Forced expression of miR-375 in EC cell lines resulted in decreased ERBB2 and VEGFA expression. C. Map of the plasmids pEZX-MT04 containing miR-375, and pEZX-MT05 containing 3'-UTR of ERBB2 to illustrate the binding site of miR-375 at the 3'-UTR of ERBB2, and its mutant control sequence. D. Dual luciferase reporter assay. Co-transfection with pEZX-miR-375 and pEZX-ERBB2 3' UTR wild type significantly decreased the luciferase activities compared to that with pEZX-miR-375 and ERBB2 3' UTR mutant/miR-375 scrambled control and ERBB2 3' UTR wild type sequence in ESCC cell lines. The data were reported as mean +- S.D. from three independent experiments (** p < 0.01, *p

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Experimental details: Figure 5 Representative western blotting showing estrogen receptor alpha, estrogen receptor beta, cyclin D1, caspase 3 (cleaved form) in Apc Min/+ mice assuming standard or enriched diet evaluated in normal and polypoid tissues; beta actin was used as loading control (panel shows representative bolts of three different experiments performed with similar results).

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Experimental details: Figure 5. miR-4463 targets cytochrome P450 family 19 subfamily A member 1 ( CYP19A1 ) and estrogen receptor alpha ( ESR1 ) genes and inhibits steroidogenesis in KGN cells. After KGN cells were transfected with a miR-4463 mimic, miR-4463 inhibitor, and miR-4463 mimic with folliclestimulating hormone (FSH), the expression levels of CYP19A1, estrogen receptor alpha (ERalpha), and synthesis of 17beta-estradiol were analyzed. (A) Expression of miR-4463 increased by knockdown of follicle-stimulating hormone receptor ( FSHR ) in KGN cells. (B) The putative sites in the 3'-untranslated region (UTR) of CYP19A1 and ESR1 mRNA contain the seed sequence for hsa-miR-4463 binding. The nucleotides in bold letters indicate the seed region of miR-4463. (C, D) Protein expression levels were measured by Western blot analysis, and beta-actin was used as an internal control. The relative protein expression levels were calculated by measuring the density and area of the bands. (E) 17beta-estradiol levels were measured by enzyme-linked immunosorbent assay using cell culture supernatant after transfection with miR-4463 mimic, miR-4463 inhibitor, and miR-4463 mimic with FSH. The experiments were repeated at least three times, and the data are presented as the mean +- standard error of the mean. siRNA, small interfering RNA. a) p < 0.05: indicate statistical significance. The p -values were obtained by one-way analysis of variance and the Student-Newman-Keuls test.

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Experimental details: Figure 6. Treatment of follicle-stimulating hormone receptor ( FSHR ) small interfering RNA (siRNA) decreased CYP19A1 expression. (A) The protein expression levels were measured by Western blot analysis, and beta-actin was used as an internal control gene. (B) The relative protein expression levels were calculated by measuring the density and area of the bands. The experiments were repeated at least three times, and the data are presented as the mean +- standard error of the mean. NC, negative control group; siFSHR, FSHR siRNA-treated group. a) p < 0.001: indicates statistical significance.

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Experimental details: Fig. 4. Microglia synaptic pruning and neuroinflammation. After IS with or without ethanol exposure for 28 days, mice were euthanized. Raphe nuclei homogenates were used to analyze protein and cytokine expression. The protein levels of Iba-1, PSD95, synaptophysin, cleaved caspase 3, and C1q were determined by Western blotting ( A ), and their intensities were semiquantified ( B ). Levels of the cytokines TNF-alpha ( C ) and IL-10 ( D ) in the raphe nuclei were determined by enzyme immunoassay. The data are expressed as the means +- SD ( n = 5). * P < 0.05 and ** P < 0.01 compared to the unstressed mice, and # P < 0.05 and ## P < 0.01 compared to the mice not exposed to ethanol. Gene expression levels of TNF-alpha ( E ) and IL-1beta ( F ) were determined by real-time PCR analysis. The data are expressed as the means +- SD ( n = 3). * P < 0.05 and ** P < 0.01 compared to the vehicle-treated cells.

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Experimental details: Figure 1 Analysis of PSMA expression levels. WB of PCa tumor tissues and a positive control, all samples were run on the same blot. The non-relevant bands were cropped from the image, the full-length blot is presented in Supplementary Fig. 1 ( a ). Semi-quantification of WB, the relative density of PSMA was normalized using beta-actin as loading control. Quantification was only performed using bands from the same blot ( b ). Data for PC-3, 22Rv1 and C4-2 (n = 5) is presented as mean +- SD. IHC of PSMA expression of PC-3 ( c ), 22Rv1 ( d ) and C4-2 ( e ) tumor tissue.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. Actin accumulates outside of the NE in prophase, and its network shrinks after NEBD. ( A ) Actin accumulates on the NE around NEBD. Images (z-projections) show a representative cell expressing GFP-tubulin and mCherry-Lifeact (that fluorescently marks F-actin). Time is relative to NEBD. Timing of NEBD was determined by the influx of cytoplasmic GFP-tubulin into the nucleus. Scale bars, 10 um. Graph shows mean Lifeact fluorescence intensity (per pixel) around the nucleus over time (n = 8; error bars, s.e.m). ( B ) Actin localizes outside of the NE. Images show single super-resolution z-sections of cells, which were fixed and stained for actin with phalloidin (actin dye; red). Cells were also immunostained (green) with anti-Lamin B1 (top) or anti-actin antibody (bottom), respectively. The same secondary antibody was used for the two immunostainings. These cells were undergoing chromosome compaction (confirmed by DNA staining). Insets show magnification of the regions in yellow boxes. Scale bar, 10 um. Graphs show intensity of actin dye (red) and immunostaining (green) along the dashed yellow lines in left images (middle parts are omitted). The peaks in graphs, marked with asterisks (top) and daggers (bottom), locate at the regions in yellow boxes in left images. a.u, arbitrary unit. ( C ) The LINC complex is required for accumulation of the actin network. Images show single z-sections of representative cells, expressing either an RFP-tagged LINC-DN construct (SR-KASH)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 6 Catalase expression in liver and the brain of fenofibrate-treated animals. Catalase levels were determined by western blot in brain and liver of ethanol or saccharin drinking rats (treated with fenofibrate or vehicle; n = 7 for each group). Catalase levels were normalized against beta-actin. Bars represent the normalized densitometric quantification of the bands. Catalase expression of the saccharin group without fenofibrate administration was used as control (100%). ** p < 0.01 with respect to control; n.s. = difference statistically non-significant.