- MA5-11690 - Invitrogen Antibodies VCL antibody

Submitted references Rora Regulates Neutrophil Migration and Activation in Zebrafish.

Hsu AY, Wang T, Syahirah R, Liu S, Li K, Zhang W, Wang J, Cao Z, Tian S, Matosevic S, Staiger CJ, Wan J, Deng Q
Frontiers in immunology 2022;13:756034

The Downregulation of Both Giant HERCs, HERC1 and HERC2, Is an Unambiguous Feature of Chronic Myeloid Leukemia, and HERC1 Levels Are Associated with Leukemic Cell Differentiation.

Ali MS, Magnati S, Panuzzo C, Cilloni D, Saglio G, Pergolizzi B, Bracco E
Journal of clinical medicine 2022 Jan 10;11(2)

The RabGAPs EPI64A and EPI64B regulate the apical structure of epithelial cells (†).

Miller MR, McDermitt DJ, Sauvanet C, Lombardo AT, Zaman R, Bretscher A
Molecular biology of the cell 2022 Jan 1;33(1):ar8

Effect of Epigallocatechin-3-Gallate on EGFR Signaling and Migration in Non-Small Cell Lung Cancer.

Minnelli C, Cianfruglia L, Laudadio E, Mobbili G, Galeazzi R, Armeni T
International journal of molecular sciences 2021 Oct 31;22(21)

Flavonoids and Omega3 Prevent Muscle and Cardiac Damage in Duchenne Muscular Dystrophy Animal Model.

Tripodi L, Molinaro D, Farini A, Cadiao G, Villa C, Torrente Y
Cells 2021 Oct 28;10(11)

IL-9 Abrogates the Metastatic Potential of Breast Cancer by Controlling Extracellular Matrix Remodeling and Cellular Contractility.

Das S, Surve V, Marathe S, Wad S, Karulkar A, Srinivasan S, Dwivedi A, Barthel SR, Purwar R
Journal of immunology (Baltimore, Md. : 1950) 2021 Jun 1;206(11):2740-2752

The Giant HECT E3 Ubiquitin Ligase HERC1 Is Aberrantly Expressed in Myeloid Related Disorders and It Is a Novel BCR-ABL1 Binding Partner.

Ali MS, Panuzzo C, Calabrese C, Maglione A, Piazza R, Cilloni D, Saglio G, Pergolizzi B, Bracco E
Cancers 2021 Jan 19;13(2)

Defective dystrophic thymus determines degenerative changes in skeletal muscle.

Farini A, Sitzia C, Villa C, Cassani B, Tripodi L, Legato M, Belicchi M, Bella P, Lonati C, Gatti S, Cerletti M, Torrente Y
Nature communications 2021 Apr 8;12(1):2099

PTX3 Predicts Myocardial Damage and Fibrosis in Duchenne Muscular Dystrophy.

Farini A, Villa C, Di Silvestre D, Bella P, Tripodi L, Rossi R, Sitzia C, Gatti S, Mauri P, Torrente Y
Frontiers in physiology 2020;11:403

Lysine in Combination With Estradiol Promote Dissemination of Estrogen Receptor Positive Breast Cancer via Upregulation of U2AF1 and RPN2 Proteins.

Vazquez Rodriguez G, Abrahamsson A, Turkina MV, Dabrosin C
Frontiers in oncology 2020;10:598684

LHRH-Conjugated Drugs as Targeted Therapeutic Agents for the Specific Targeting and Localized Treatment of Triple Negative Breast Cancer.

Obayemi JD, Salifu AA, Eluu SC, Uzonwanne VO, Jusu SM, Nwazojie CC, Onyekanne CE, Ojelabi O, Payne L, Moore CM, King JA, Soboyejo WO
Scientific reports 2020 May 19;10(1):8212

Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression.

Lim KH, Han Z, Jeon HY, Kach J, Jing E, Weyn-Vanhentenryck S, Downs M, Corrionero A, Oh R, Scharner J, Venkatesh A, Ji S, Liau G, Ticho B, Nash H, Aznarez I
Nature communications 2020 Jul 9;11(1):3501

Truncation of MYH8 tail in AML: a novel prognostic marker with increase cell migration and epithelial-mesenchymal transition utilizing RAF/MAPK pathway.

Park H, Kim D, Kim D, Park J, Koh Y, Yoon SS
Carcinogenesis 2020 Jul 10;41(6):817-827

Transferrin-targeted porous silicon nanoparticles reduce glioblastoma cell migration across tight extracellular space.

Sheykhzadeh S, Luo M, Peng B, White J, Abdalla Y, Tang T, Mäkilä E, Voelcker NH, Tong WY
Scientific reports 2020 Feb 11;10(1):2320

Vinculin variant M94I identified in sudden unexplained nocturnal death syndrome decreases cardiac sodium current.

Cheng J, Kyle JW, Wiedmeyer B, Lang D, Vaidyanathan R, Makielski JC
Scientific reports 2017 Feb 20;7:42953

An East Asian Common Variant Vinculin P.Asp841His Was Associated With Sudden Unexplained Nocturnal Death Syndrome in the Chinese Han Population.

Cheng J, Kyle JW, Lang D, Wiedmeyer B, Guo J, Yin K, Huang L, Vaidyanathan R, Su T, Makielski JC
Journal of the American Heart Association 2017 Apr 3;6(4)

EGFR Interacts with the Fusion Protein of Respiratory Syncytial Virus Strain 2-20 and Mediates Infection and Mucin Expression.

Currier MG, Lee S, Stobart CC, Hotard AL, Villenave R, Meng J, Pretto CD, Shields MD, Nguyen MT, Todd SO, Chi MH, Hammonds J, Krumm SA, Spearman P, Plemper RK, Sakamoto K, Peebles RS Jr, Power UF, Moore ML
PLoS pathogens 2016 May;12(5):e1005622

Focal adhesion plaque associated cytoskeletons are involved in the invasion and metastasis of human colorectal carcinoma.

Yang HJ, Chen JZ, Zhang WL, Ding YQ
Cancer investigation 2010 Feb;28(2):127-34

Connective tissue growth factor expression and induction by transforming growth factor-beta is abrogated by simvastatin via a Rho signaling mechanism.

Watts KL, Spiteri MA
American journal of physiology. Lung cellular and molecular physiology 2004 Dec;287(6):L1323-32

Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.

Willey CD, Balasubramanian S, Rodríguez Rosas MC, Ross RS, Kuppuswamy D
Journal of molecular and cellular cardiology 2003 Jun;35(6):671-83

Focal complex formation in adult cardiomyocytes is accompanied by the activation of beta3 integrin and c-Src.

Willey CD, Balasubramanian S, Rodríguez Rosas MC, Ross RS, Kuppuswamy D
Journal of molecular and cellular cardiology 2003 Jun;35(6):671-83

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Supportive validation

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Experimental details: Western blot of Vinculin using Vinculin Monoclonal Antibody (Product # MA5-11690) on HeLa Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of HeLa (Lane 1), NIH/3T3 (Lane 2), COS-7 (Lane 3), Hep G2 (Lane 4), Caco-2 (Lane 5), HT-29 (Lane 6), T-47D (Lane 7), K-562 (Lane 8), U-2 OS (Lane 9), HEK-293 (Lane 10). The blots were probed with Anti-Vinculin Mouse Monoclonal Antibody (Product # MA5-11690, 1-3 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conj µgate (Product # A28177, 0.4 µg/mL, 1:2500 dilution). A 124 kDa band corresponding to Vinculin was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10 % Bis-Tris gel (Product # NP0302BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with overnight wet transfer system. The membrane was probed with the relevant primary and secondary Antibody using iBind™ Flex Western Starter Kit (Product # SLF2000S). Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of T-47D (Lane 1),K-562 (Lane 2), L6 (Lane 3), C2C12 (Lane 4), COS-7 (Lane 5), MOLT4 (Lane 6), Ramos (Lane 7) and Jurkat (Lane 8). The blot was probed with Vinculin Mouse Monoclonal Antibody (Product # MA5-11690, 1:250 dilution) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). A 124 kDa band corresponding to Vinculin was observed in T-47D, K-562, L6, C2C12, COS-7 and not observed in other cell lines which are documented to be Vinculin negative.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Vinculin was performed using 70% confluent log phase U-2 OS cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were labeled with Vinculin (VLN01) Mouse Monoclonal Antibody (Product # MA5-11690) at 2 µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conj µgate (Product # A28175) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing cytoplasmic localization. Panel e shows the no primary antibody control. The images were captured at 60X magnification.

Supportive validation

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Experimental details: Flow cytometry analysis of Vinculin was done on K-562 cells. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 30 minutes at room temperature. Cells were labeled with Vinculin Mouse Monoclonal Antibody (Product # MA5-11690, red histogram) or with mouse isotype control (pink histogram) at 3-5 µg/million cells in 2.5% BSA. After incubation at room temperature for 2 hours, the cells were labeled with Alexa Fluor® 488 Rabbit Anti-Mouse Secondary Antibody (Product # A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10, 000 cells were acquired and analyzed for each sample using an Attune® Acoustic Focusing Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunoprecipitation of Vinculin using Vinculin Monoclonal Antibody (Product # MA5-11690) on denatured Human LS174T Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 1 Pentraxin (PTX)3 expression in skeletal muscle biopsies of mdx mice at different ages. Representative Western blot (WB) of PTX3 in TAs of 10 weeks (10w), 3 months (3m), 5m, 7m C57Bl mice and in TAs of 10w, 9m, 14m, and 18m mdx mice. Data from densitometric analysis are expressed as PTX3/vinculin ratio in arbitrary units in the lower panels. One-way ANOVA with Tukey's multiple comparisons test: * p < 0.05; ** p < 0.01; *** p < 0.001 (A) . In the lateral panels, the scheme representing PTX3 expression, in C57Bl and mdx mice, according to ages (B) . Representative WB of PTX3 in TA of 10w, 5m, and 9m C57Bl mice and age-matched mdx mice. Data from densitometric analysis are expressed as the ratio of PTX3/vinculin in arbitrary units in the lateral panels. Student's t -test: * p < 0.05; ** p < 0.01 (C) . Each experiment was performed in triplicate wells. All values are expressed as the mean +- SD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 2 Pentraxin (PTX)3 expression in skeletal cardiac tissues of mdx mice at different ages. Representative Western blot (WB) of PTX3 in cardiac tissues of 10 weeks (10w) and 5 months (5m) C57Bl mice and age-matched mdx mice. Data from densitometric analysis are expressed as the ratio of PTX3/vinculin in arbitrary units in lateral panels. One-way ANOVA with Tukey's multiple comparisons test: * p < 0.05 (A) . Representative WB of PTX3 in cardiac tissues of 11 days (11dy), 10w, 9m, and 18m mdx mice. Data from densitometric analysis are expressed as PTX3/vinculin ratio in arbitrary units in the lateral panel. One-way ANOVA with Tukey's multiple comparisons test: * p < 0.05; **** p < 0.0001 (B) . The line graph represents the dependence of PTX3 expression according to the age in mdx cardiac tissues (C) . ELISA quantification of PTX3 in cardiac muscles of 10w C57Bl and 10w and 9m mdx mice related to skeletal muscles of age-matched mice. Student's t -test: * p < 0.05; *** p < 0.001; **** p < 0.0001 (D) . (E) Representative images of hearts from 10w C57Bl and 10w, 9m, and 18m mdx mice ( n = 5 each) showing PTX3 and vascular staining. Representative images of PTX3 immunohistochemistry staining of endothelial (arrowheads) and mural cells (pericytes) (arrows) within coronary vessel wall from C57Bl and mdx (first row). Representative images of coronary vessels expressing smooth muscle actin (SMA) (green) and CD31 (red) showing increased expression of NG2 (magenta) and altered morphol

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 5 Western blot (WB) analysis of immunoproteasome (IP) subunits and alarmins in cardiac tissues of mdx mice at different ages. Representative WB of PSMB5, PSMB8, PSMB9, pERK/total ERK, and p38/total p38 in cardiac tissues of 11 days (11dy), 10 weeks (10w), 9 months (9m), and 18m mdx mice (A) . In the lateral panel, densitometric analysis of data, expressed as the ratio of different proteins versus vinculin in arbitrary units. One-way ANOVA with Tukey's multiple comparisons test: * p < 0.05 (B) . Representative WB of receptor for advanced glycation end-products (RAGE), S100beta, Foxp3, monocyte chemoattractant protein (MCP)-1, high-mobility group box (HMGB)1, and interleukin (IL)-33 in cardiac tissues of 11dy, 10w, 9m, and 18m mdx mice (C) . Data from densitometric analysis are expressed as the ratio of different proteins versus vinculin in arbitrary units in the lateral panel (D) . One-way ANOVA with Tukey's multiple comparisons test: * p < 0.05; ** p < 0.01; *** p < 0.001. Each experiment was performed in triplicate wells. All values are expressed as the mean +- SD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 6 Evaluation of proteins involved in inflammation and fibrosis in cardiac tissues of mdx mice at different ages. Representative Western blot (WB) of several inflammatory and fibrotic mediators in cardiac tissues of 11 days (11dy), 10 weeks (10w), 9 months (9m), and 18m mdx mice (A) . Data from densitometric analysis are expressed as the ratio of different proteins versus vinculin in arbitrary units in the lateral panel. One-way ANOVA with Tukey's multiple comparisons test: * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 (B) . Each experiment was performed in triplicate wells. All values are expressed as the mean +- SD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 7 Evaluation of inflammatory cytokines and autophagic mediators in cardiac tissues of mdx mice at different ages. Representative Western blot (WB) of transforming growth factor (TGF)-b, poly(ADP-ribose) polymerase (PPAR)g, interleukin (IL)-6, and tumor necrosis factor (TNF)-a in cardiac tissues of 11 days (11dy), 10 weeks (10w), 9 months (9m), and 18m mdx mice (A) . Data from densitometric analysis are expressed as the ratio of different proteins versus vinculin in arbitrary units in the lateral panel. One-way ANOVA with Tukey's multiple comparisons test: * p < 0.05; ** p < 0.01; *** p < 0.001 (B) . Representative WB of ATG-7, LC3B, and p62 in cardiac tissues of 11dy, 10w, 9m, and 18m mdx mice (C) . Data from densitometric analysis are expressed as the ratio of different proteins versus vinculin in arbitrary units in the lateral panels. One-way ANOVA with Tukey's multiple comparisons test: ** p < 0.01; *** p < 0.001 (D) . Each experiment was performed in triplicate wells. All values are expressed as the mean +- SD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 8 Modulation of proteins in cardiac tissues of mdx mice at different ages following ONX-0914 treatment. Representative images of pentraxin (PTX)3 and PMSB8 immunohistochemistry staining of cardiac tissues showing PTX3- and PSMB8-positive endothelial cells (arrowheads) in cardiac tissues from 9 months (9m) and 18m mdx mice. Image magnifications: 10 x (scale bar: 200 mm) and 40 x (scale bar: 50 mm) (A) . Representative Western blot (WB) in cardiac tissues of untreated and ONX-0914-treated 9m mdx mice for Toll-like receptor (TLR)2, TLR4, PTX3 (B) ; receptor for advanced glycation end-products (RAGE), high-mobility group box (HMGB)1, S100beta, and interleukin (IL)-33; collagen I, collagen VI, matrix metalloproteinase (MMP)9, and TRAF-6 (C) ; tumor necrosis factor (TNF)-a, poly(ADP-ribose) polymerase (PPAR)g, MYD88, RelB, nuclear factor (NF)-kappaB, ATG-7, and LC3B (D) . Data from densitometric analysis are expressed as the ratio of different proteins versus vinculin (B) and actin (C,D) in arbitrary units in the lateral panels. Student's t -test: ** p < 0.01; *** p < 0.001 (B) ; * p < 0.05 (C) ; * p < 0.05; *** p < 0.001 (D) . Each experiment was performed in triplicate wells. All values are expressed as the mean +- SD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 Confocal fluorescence images showing cellular uptake and cytotoxicity comparison of MDA-MB-231 cells 6 hours after their incubation with 30 muM of PGS, PGS-LHRH, PTX or PTX-LHRH (arrows indicate the structural changes in the nuclei structure (blue), actin cytoskeleton structure (red) and vinculin structure (green).

Supportive validation

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Experimental details: Figure 4 Knockdown of U2AF1 decreased luminal A BC cell dissemination in vivo . Luminal A ER+ MCF-7 cells, transfected with negative control siRNA (siRNA-C) or siRNA targeting U2AF1 (siRNA-U2AF1) were injected in presence of estradiol (E2) +- neutrophils (Neu) into zebrafish transgenic embryos, with green fluorescent blood vessels, and analyzed as described in materials and methods. (A) Migration in vitro (n = 6-12). (B) In vivo dissemination in presence of E2 +- Neu (n = 38-41). Scale bar = 100 um. (C) Western blot analysis for confirmation of siRNA-U2AF1 transfection and ICAM-1, VCAM-1, and MUC-1 expression. (D) Focal adhesion area (n = 7). Scale bar = 10 um. (E) Proliferation in vitro (n = 12). Representative images of zebrafish embryos with disseminated MCF-7 cells and immunocytochemistry analysis of vinculin expression are shown. Arrows show disseminated MCF-7 cells and arrowheads show focal adhesions. BV = blood vessels. Data are presented as mean +- SEM. Two-tailed Student's t-test *P < 0.05, **P < 0.01, ns, not significant. Data are represented of at least two independent experiments.

Supportive validation

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Experimental details: Figure 6 Knockdown of RPN2 decreased luminal B BC cell dissemination in vivo . Luminal B ER+ T47D cells, transfected with negative control siRNA (siRNA-C) or siRNA targeting RPN2 (siRNA-RPN2) were injected + estradiol (E2) +- neutrophils (Neu) into zebrafish transgenic embryos with green fluorescent blood vessels and analyzed as described in materials and methods. (A) Migration in vitro (n = 6). (B) In vivo dissemination of transfected T47D in presence of E2 +- Neu (n = 23-26). Scale bar = 100 um. (n = 23-26). (C) Western blot analysis for confirmation of siRNA-RPN2 transfection and VCAM-1, ICAM-1, and MUC-1 expression. (D) Focal adhesions area (n = 5-6). Scale bar = 10 um. (E) Proliferation (n = 6). Representative images of zebrafish embryos with disseminated luminal B T47D BC cells and immunocytochemistry analysis of vinculin expression are shown. Arrows show disseminated T47D and arrowheads show focal adhesions. BV = blood vessels. Data are presented as mean +- SEM. Two-tailed Student's t-test *P < 0.05, ***P < 0.001, ns, not significant. Data are represented of at least two independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 6 Adult dystrophic thymus transplantation determines dystrophic muscle features and skeletal muscle regression. Representative immune fluorescence staining with DYS-2 (C-terminal-domain) antibody. Overall, confocal microscope images of TAs from nu PBS , Tnu MDX and Tnu C57Bl showed weak dystrophin intensity around the myofibers in TA of Tnu MDX mice ( a ). Overview and higher magnification of ATPase (pH 4.3) muscle sections of TAs from nu PBS , Tnu MDX and Tnu C57Bl mice ( a ). Densitometric analysis of WB images of dystrophin protein expression showed downregulation of Dp427 dystrophin isoform in TA muscles of Tnu MDX and Tnu C57Bl mice ( b ). RT-PCR analysis of TA of nu PBS , Tnu MDX and Tnu C57Bl mice determined the expression of Dp427 dystrophin isoform ( c ). Representative images of skeletal muscle showed the distribution and composition of the myosin heavy chain (MyHC) isoforms (Type IIa, Type IIx and Type IIb). Graph portrays the percentage of myofibers expressing different MyHC isoforms in TAs of nu PBS , Tnu MDX and Tnu C57Bl mice. n = 10 images were analysed for each mouse ( d ). RT-qPCR experiments on TA muscles demonstrated the over-expression of MyHC-sl2 gene together with the downregulation of fast atp2a1 in TA of Tnu MDX mice related to TAs of other mice ( e ). Tnu MDX mice showed a dramatic weight loss ( f ), which correlated with the over-expression of the atrophy-related MuRF-1 gene ( g ). Cropped image of a representative WB showing the expression of

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 VCL directly interacts with SCN5A. ( A ) Mouse liver and heart (HT) tissue lysates were immunoprecipitated (IP) using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies; ( B ) Wild-type VCL (VCL-WT) and SCN5A were transfected into HEK293 cells for 24 h, cell lysates were IP using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies; ( C ) Mutant VCL (VCL-M94I) and SCN5A were transfected into HEK293 cells for 24 h, cell lysates were IP using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies; ( D ) VCL-WT, VCL-M94I and SCN5A were transfected into HEK293 cells for 24 h, cell lysates were IP using VCL or SCN5A antibody and analyzed by Western blotting using the indicated antibodies. The results are representative of three independent experiments.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5 The effect of EGCG on NSCLC cell migration. ( A ) A549, ( B ) HCC827, and ( C ) H1975 cell migration after different EGCG treatments (30, 70, and 90 uM) was evaluated by scratch wound assay. Images were taken at times 0, 48, and 72 h. The wound area was measured by ImageJ software. The percentage migration was calculated by the average area reduction at 24, 48, and 72 h as compared to time 0. The red dotted box represents the size of the original wound. The data is the mean of triplicate experiments +- SD. Scale bar: 100 mum. ( D ) Representative Western blot experiment depicting total vinculin and metavinculin expression after 24 h of EGCG treatment. ( E ) Densitometric analysis showed differences in vinculin expression. GAPDH was used as an internal loading control. * p < 0.05; ** p < 0.01. GAPDH is the same as Figure 4 D as the blots were derived from the same membrane incubated with different antibodies. Full-length blots are presented in Supplementary Figure S1 . The samples were derived from the same experiment and the blots were processed in parallel.

Supportive validation

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Experimental details: Figure 4 Effect of Imatinib and Dasatinib treatment on HERC1 and HERC2 expression in primary CML and K562 (Ph+) cells. The HERC1 and HERC2 gene expressions following Imatinib (1 muM) and Dasatinib (0.25 uM) treatment were determined at mRNA and protein level by RT-qPCR ( A , C , E , F ), immunofluorescence ( B , D ), and Western Blotting ( G ). Imatinib (1 uM) and Dasatinib (0.25 uM) treated K-562 cells (24 h) showed an increase in HERC1 gene expression both at mRNA and protein levels compared to non-treated cells, in contrast, an increase in HERC2 mRNA was observed without any change in its protein level. TKIs impaired the phosphorylation (enzymatic actively) of BCR-ABL, which indicates the HERC1 protein and mRNA levels of both HERCs are modulated by the activity of Bcr-Abl1. Vinculin was used as a loading control in Western blot. p

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 8: Caco-2 cells lacking EPI64A and EPI64B have aberrant apical junctions (A) Fields of wild-type, EPI64A and EPI64B single knockout and EPI64A/B double knockout cells stained for actin and the tight junction marker ZO-1. The phenotypes seen were variable, so the most wild-type-looking regions of cells are shown (Normal) and contrasted with regions where the normal polygonal organization is disrupted (Severe). Scale bar: 10 um. (B) Percentage of wild-type and knockout cells in which one or more of its junctions shows a reflex angle (>180deg). One-way analysis of variance gave the indicated p values. (C) Example of stellate knockout cell stained for ezrin, myosin IIA, and actin XY-dimensions (top panels) and YZ-dimensions (bottom panel). (D) Localization of vinculin, actin, and myosin IIA in the apical (top panels) and basal (bottom panels) sections of wild-type and double knockout Caco-2 cells. Scale bars 10 um.

MA5-11690

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