antibodies against integrin α6  (Bio-Rad)

Journal: Aging (Albany NY)

Article Title: Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin β4 axis

doi: 10.18632/aging.103425

Figure Lengend Snippet: ANPEP expression was downregulated upon cell suspension. ( A ) The expression level of ANPEP in adherent, suspended, and re-attached melanoma cells as examined by qPCR and flow cytometry. Downregulation of ANPEP at the surface of suspended or re-attached melanoma cells was observed. Data were mean ±S.D. (n=3); **, p < 0.01. ( B ) Expression of integrin isoforms in adherent and suspended melanoma cells as examined by qPCR. Data were mean ±S.D. (n=3); *, p < 0.05; **, p < 0.01. ( C ) Integrin α6, β2, and β4 protein expression upon cell suspension as examined by western blot.

Article Snippet: The primary antibodies against integrin α6 and integrin β2 were from Bioss antibodies Inc. (Woburn, Massachusetts, USA).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin β4 axis

doi: 10.18632/aging.103425

Figure Lengend Snippet: Integrin β4 expression was downregulated upon suppression of SDC1 expression. ( A ) Effect of SDC1 downregulation at expression of integrin isoforms as examined by qPCR. Data were mean ±S.D. (n=3); **, p < 0.01. ( B ) Integrin α6, β2, and β4 protein expression after SDC1 downregulation as examined by western blot. ( C ) SDC1 downregulation by shSDC1 did not change the level of ANPEP expression as examined by qPCR. Data were mean ±S.D. (n=3).

Article Snippet: The primary antibodies against integrin α6 and integrin β2 were from Bioss antibodies Inc. (Woburn, Massachusetts, USA).

Techniques: Expressing, Western Blot

Journal: Aging (Albany NY)

Article Title: Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin β4 axis

doi: 10.18632/aging.103425

Figure Lengend Snippet: ANPEP regulated SDC1 and integrin β4 expression through PKCδ phosphorylation. ( A ) Suppression of ANPEP expression by ANPEP-specific shRNAs and were examined by qPCR and flow cytometry. Data were mean ±S.D. (n=3); **, p < 0.01. ( B ) Effect of ANPEP downregulation at expression of SDC1 and integrin isoforms as examined by qPCR. Data were mean ±S.D. (n=3); **, p < 0.01. ( C ) Effect of ANPEP downregulation at expression of integrin isoforms, SDC1, and PKCδ phosphorylation as examined by western blot.

Article Snippet: The primary antibodies against integrin α6 and integrin β2 were from Bioss antibodies Inc. (Woburn, Massachusetts, USA).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Aging (Albany NY)

Article Title: Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin β4 axis

doi: 10.18632/aging.103425

Figure Lengend Snippet: ANPEP was tumor suppressive in melanoma cells. ( A ) Suppression of ANPEP expression inhibited cell migration ability ( B ) Suppression of ANPEP expression promoted xenograft tumor growth as inoculated with adherent melanoma cells. ( C ) Effect of ANPEP overexpression at PKCδ phosphorylation, SDC1, and integrin β4 protein expression as examined by western blot. ( D ) ANPEP overexpression reduced the xenograft tumor formation as inoculated with suspended melanoma cells Data were mean ±S.D. (n=5).

Article Snippet: The primary antibodies against integrin α6 and integrin β2 were from Bioss antibodies Inc. (Woburn, Massachusetts, USA).

Techniques: Expressing, Migration, Over Expression, Western Blot

Journal: Aging (Albany NY)

Article Title: Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin β4 axis

doi: 10.18632/aging.103425

Figure Lengend Snippet: Immunostaining showed different vascular phenotypes with different protein expressions upon cell suspension and suppression of ANPEP expression. Photomagnification of tumor tissues derived from ( A ) adherent melanoma cells or ( C ) shLuc-transfected cells with HE stain and immunostaining of CD31 showed vascular structures. Positive ANPEP, SDC1, and integrin β4 expression in these tumor tissues were also presented. Filled arrow, vascular cavities with integrin β4-positive cells-surrounded. Empty arrow, vascular cavities with integrin β4-positive cells. No vascular structures with CD31/ANPEP/SDC1/integrin β4 expression were seen in those from ( B ) suspended melanoma cells or ( D ) shANPEP-transfected cells. The arrows indicated the locations of RBC-infiltrated cavities.

Article Snippet: The primary antibodies against integrin α6 and integrin β2 were from Bioss antibodies Inc. (Woburn, Massachusetts, USA).

Techniques: Immunostaining, Expressing, Derivative Assay, Transfection, H&E Stain

Journal: Aging (Albany NY)

Article Title: Anchorage independence altered vasculogenic phenotype of melanoma cells through downregulation in aminopeptidase N /syndecan-1/integrin β4 axis

doi: 10.18632/aging.103425

Figure Lengend Snippet: Scheme for ANPEP-mediated downregulation of SDC1 and integrin β4 mediated by PKCδ activation, so that contributed to the loss of vasculogenic phenotypes.

Article Snippet: The primary antibodies against integrin α6 and integrin β2 were from Bioss antibodies Inc. (Woburn, Massachusetts, USA).

Techniques: Activation Assay

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: USP33 is an integrin α6 deubiquitinase and promotes esophageal squamous cell carcinoma cell migration and metastasis

doi: 10.1007/s00432-024-06041-5

Figure Lengend Snippet: USP33 regulates integrin α6 stability via deubiquitination. ( A ) Cell lysates from USP33-knockdown KYSE-150 cells were harvested and then immunoblotted with antibodies against integrin β1, integrin β4, integrin α3, integrin α5, integrin α6, USP33, and GAPDH. ( B ) KYSE-150 cell lysates were collected and immunoprecipitated using an anti-USP33 antibody; the immunoprecipitants were immunoblotted with antibodies against integrin β4, integrin α6, and USP33. ( C ) KYSE-150 cells were co-transfected with HA-ubiquitin (Ub) and USP33 or GFP, followed by denature immunoprecipitation through anti-integrin α6 antibody and then immunoblotting with antibodies against integrin α6 and HA

Article Snippet: Mouse mAbs against integrin α6 (1:500, Catalog #sc-374057) and protein-A/G PLUS beads for immunoprecipitation, GAPDH (1:500, Catalog #sc-365062), integrin α3 (1:500, Catalog #sc-374242), and β-actin (1:500, Catalog #sc-47778) for WB were from Santa Cruz Biotechnology (Texas, USA).

Techniques: Knockdown, Immunoprecipitation, Transfection, Ubiquitin Proteomics, Western Blot

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: USP33 is an integrin α6 deubiquitinase and promotes esophageal squamous cell carcinoma cell migration and metastasis

doi: 10.1007/s00432-024-06041-5

Figure Lengend Snippet: USP33 promotes laminin-mediated cell migration and metastasis through integrin α6. ( A and B ) Cell lysates were harvested from the Con, USP33-sh#1, USP33-sh#1 + USP33, USP33-sh#1 + Integrin α6 KYSE-150 ( A ) and KYSE-450 ( B ) cell groups, and then immunoblotted with antibodies against USP33, integrin α6, and β-actin. ( C ) Transwell migration assay of indicated cells described in ( A and B ) on laminin-coated condition. ( n = 3 biological replicates, one-way ANOVA test). ( D ) H&E staining images and quantification of the number of metastatic nodules in the lungs from BALB/c-nude mice following tail vein injection of Con, USP33-sh#1, USP33-sh#1 + USP33, USP33-sh#1 + Integrin α6 KYSE-150 cells (Scale bar, 200 μm; n = 5, one-way ANOVA)

Article Snippet: Mouse mAbs against integrin α6 (1:500, Catalog #sc-374057) and protein-A/G PLUS beads for immunoprecipitation, GAPDH (1:500, Catalog #sc-365062), integrin α3 (1:500, Catalog #sc-374242), and β-actin (1:500, Catalog #sc-47778) for WB were from Santa Cruz Biotechnology (Texas, USA).

Techniques: Migration, Transwell Migration Assay, Staining, Injection

Journal: Journal of Mammary Gland Biology and Neoplasia

Article Title: Spatial Engineering of Mammary Epithelial Cell Cultures with 3D Bioprinting Reveals Growth Control by Branch Point Proximity

doi: 10.1007/s10911-024-09557-1

Figure Lengend Snippet: Extrusion 3D bioprinting of mammary epithelial cells inside an ECM leads to formation of polarized 3D cell cultures of a defined shape. A-B Immunofluorescence imaging of the BM protein laminin α5 (LAMA5, green) and nuclei (blue) in clonal spheroid cultures of MCF10A cells A , and 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells B on day 14. Magnified images are shown in the ROIs. The MCF10A spheroids embedded in an ECM with elevated BME content A produced results comparable to the ECM used in B . C-D Immunofluorescence imaging of the basal epithelial integrin α6 (ITGA6, green) and nuclei (blue) in clonal spheroid cultures of MCF10A cells C , and 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells D on day 14. Magnified images are shown in the ROIs. The MCF10A spheroids embedded in an ECM with elevated BME content C produced results comparable to the ECM used in D . E Immunofluorescence imaging of E-cadherin (green), vimentin (magenta) and nuclei (blue) in 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells on day 10. Magnified images are shown in the ROIs. F Immunofluorescence imaging of nuclei (DAPI) in the 3D-bioprinted cultures of MCF10A and MCF10DCIS.com cells on day 14. Some of the apoptotic cells with fragmented nuclei are indicated with yellow arrowheads. Scale bars A, C: 100 μm, ROI 25 μm; B, D, E: 500 μm, ROI 50 μm; F: 50 μm. Images represent the central plane of the cell cultures

Article Snippet: The gels were incubated with primary antibodies against integrin α6 (NKI-GoH3, MCA699GA, Bio-Rad, 1:800), laminin α5 (4C7, ab17107-1001, Abcam, 1:50), E-cadherin (24E10, 3195 S, Cell Signaling, 1:200), vimentin (V9, 347 M-1, Sigma, 1:1000) and Ki67 (GR3375556-1, ab15580, Abcam, 1:250) in the blocking solution for 3 days at 4 °C on a shaker.

Techniques: Immunofluorescence, Imaging, Produced

Journal: Oncogenesis

Article Title: Decreased B4GALT1 promotes hepatocellular carcinoma cell invasiveness by regulating the laminin-integrin pathway.

doi: 10.1038/s41389-023-00494-y

Figure Lengend Snippet: Fig. 4 B4GALT1 regulates the glycosylation of integrins α6 and β1 in HCC cells. A GSL-II lectin pull-down assay for integrin β1. Proteins in cell lysates from B4GALT1 knockdown PLC5 and HA22T cells and B4GALT1 knockout PLC5 cells were pulled down using GSL-II agarose beads and immunoblotted using an anti-integrin β1 (ITGB1). Three independent siRNAs for B4GALT1and two independent B4GALT1 knockout clones were used, as indicated. B GSL-II lectin pull-down assay for integrin α6. Proteins in the cell lysates from B4GALT1 knockdown PLC5 and HA22T cells and B4GALT1 knockout PLC5 cells were pulled down using GSL-II agarose beads and immunoblotted using an anti-integrin α6 (ITGA6). C B4GALT1 predominantly modified N-glycans on integrin β1. PLC5 cell lysates (0.5 mg) were treated with PNGase F and then pulled down (PD) using GSL-II lectin. The pull-down proteins were separated using 6% SDS-PAGE and analyzed via immunoblotting with anti-ITGB1. D B4GALT1 predominantly modified N-glycans on integrin α6. PLC5 cell lysates (0.5 mg) were treated with PNGase F and then pulled down (PD) using GSL-II lectin. The pulled-down proteins were separated using 6% SDS-PAGE and analyzed via immunoblotting using anti-ITGA6 (A) or anti-ITGA6 (B) antibody. Representative results from three independent experiments are shown. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t-test.

Article Snippet: Further, antibody against integrin α6 was obtained from Cell Signaling Technology Inc. (Cat#3750), while that against GAPDH was purchased from Meridian Life Science. (Cat#H86504M).

Techniques: Glycoproteomics, Pull Down Assay, Knockdown, Knock-Out, Clone Assay, SDS Page, Western Blot

Journal: Oncogenesis

Article Title: Decreased B4GALT1 promotes hepatocellular carcinoma cell invasiveness by regulating the laminin-integrin pathway.

doi: 10.1038/s41389-023-00494-y

Figure Lengend Snippet: Fig. 6 Blockade of integrin β1 or integrin α6 inhibits migration and invasion promoted by B4GALT1 knockdown or knockout in HCC cells. A Transwell migration assays showing the effect of anti-integrin β1 antibody. The increased cell migration increased by B4GALT1 siRNA in HA22T and PLC5 cells, or B4GALT1 knockout (ko) in PLC5 cells was significantly blocked by an anti-integrin β1 (ITGB1) antibody. Two different B4GALT1 siRNAs and one non-targeting control siRNA were used for HA22T and PLC5 cells. Two independent clones of B4GALT1 knockout PLC5 cells and wild-type PLC5 cells were used. Mouse IgG was used as control. B Transwell migration assays showing the effect of anti-integrin α6 antibody. The increased cell migration by B4GALT1 siRNA in HA22T and PLC5 cells, or B4GALT1 knockout (ko) in PLC5 cells was significantly blocked by anti-integrin α6 (ITGA6) antibody. C Matrigel invasion assays showing the effect of anti-integrin β1 antibody. The increased cell invasion increased by B4GALT1 siRNA in HA22T and PLC5 cells, or B4GALT1 knockout (ko) in PLC5 cells was significantly blocked by anti-ITGB1 antibody. D Matrigel invasion assays showing the effect of anti-integrin α6 antibody. The increased cell invasion increased by B4GALT1 siRNA in HA22T and PLC5 cells, or B4GALT1 knockout (ko) in PLC5 cells was significantly blocked by anti-ITGA6 antibody. Representative results from three independent experiments are shown. Data are presented as the mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t-test.

Article Snippet: Further, antibody against integrin α6 was obtained from Cell Signaling Technology Inc. (Cat#3750), while that against GAPDH was purchased from Meridian Life Science. (Cat#H86504M).

Techniques: Migration, Knockdown, Knock-Out, Control, Clone Assay

Journal: Oncogenesis

Article Title: Decreased B4GALT1 promotes hepatocellular carcinoma cell invasiveness by regulating the laminin-integrin pathway.

doi: 10.1038/s41389-023-00494-y

Figure Lengend Snippet: Fig. 7 A schematic diagram illustrating the proposed mechanism by which B4GALT1 regulates HCC invasiveness. Downregulation of B4GALT1 promotes the invasive behaviors of HCC through the alteration of N-glycosylation and function of the laminin receptor, integrin α6β1.

Article Snippet: Further, antibody against integrin α6 was obtained from Cell Signaling Technology Inc. (Cat#3750), while that against GAPDH was purchased from Meridian Life Science. (Cat#H86504M).

Techniques: Glycoproteomics

Journal: BMC Cell Biology

Article Title: Connective Tissue Growth Factor (CTGF/CCN2) enhances lactogenic differentiation of mammary epithelial cells via integrin-mediated cell adhesion

doi: 10.1186/1471-2121-11-35

Figure Lengend Snippet: CTGF/CCN2 contributes to the stabilization of surface-level integrin complexes . ( A ) HC11-TRE and HC11-TRE-CTGF cells were incubated in serum-free media with EGF (10 ng/ml) or identical media containing either an RGD-containing peptide or RAD-containing peptide (500 μM). The MTT assay was performed at 24, 48, 72, and 96 hours. The results are displayed as the mean + the S.D. of four determinations. *, p < 0.005. ( B ) HC11-TRE and HC11-TRE-CTGF cells were grown in serum free media for 4 days in the presence of EGF (10 ng/ml). Cells were harvested and stained with an anti-β1 integrin antibody, anti-α6 antibody or isotype controls followed by detection with a fluorescence-conjugated secondary antibody and analysis by flow cytometry. The median number of β1 integrin or α6 positive cells is indicated. The data are representative of 4 independent experiments.

Article Snippet: Cells were then incubated in buffer with antibodies against β1 integrin (BD Biosciences) or α6 integrin (BD Biosciences) or the isotype controls for 1 hour at 4°C then pelleted and resuspended FACS buffer containing PE- or FITC- conjugated secondary antibodies for 45 minutes at 4°C.

Techniques: Incubation, MTT Assay, Staining, Fluorescence, Flow Cytometry

Journal: BMC Cell Biology

Article Title: Connective Tissue Growth Factor (CTGF/CCN2) enhances lactogenic differentiation of mammary epithelial cells via integrin-mediated cell adhesion

doi: 10.1186/1471-2121-11-35

Figure Lengend Snippet: CTGF/CCN2 enhances adhesion of HC11 cells through the α6β1 integrin complex . HC11-TRE cells were resuspended in serum-free growth media + EGF (10 ng/ml) and seeded on microtiter wells coated with recombinant CTGF/CCN2 (2 μg/ml) or 1% BSA wells as described in Methods. Adherent cells were fixed , stained, and quantified by reading the absorbance at 570 nm. Data are means of quadruplicate samples, error bars represent standard error. *, ◇, ■, p < 0.005. ( A ) HC11-TRE cells adhesion in serum-free growth media + EGF (10 ng/ml) with EDTA (2.5 mM), EDTA + Mg2+ (5 mM) or no addition. ( B ) HC11-TRE cells adhesion in serum-free growth media + EGF (10 ng/ml) with function-blocking antibodies or isotype controls (25 μg/ml).

Article Snippet: Cells were then incubated in buffer with antibodies against β1 integrin (BD Biosciences) or α6 integrin (BD Biosciences) or the isotype controls for 1 hour at 4°C then pelleted and resuspended FACS buffer containing PE- or FITC- conjugated secondary antibodies for 45 minutes at 4°C.

Techniques: Recombinant, Staining, Blocking Assay

Journal: Scientific reports

Article Title: Expression and distribution of CD151 as a partner of alpha6 integrin in male germ cells.

doi: 10.1038/s41598-020-61334-2

Figure Lengend Snippet: Figure 4. Localization of CD151 and integrin subunits α6 and β4 in mouse epididymal spermatozoa and their interaction. Localization of (a,b) CD151 (green), (c,d) α6 integrin (green), (e,f) α3 integrin (green), (g,h) β4 integrin (green), (i,j) β1 integrin (green) revealed by super-resolution SIM capturing. CD151 and α6, β4 integrin subunits are localized in the membrane overlying the equatorial segment of the acrosome. On the other hand, α3 and β1 integrin localization is to apical acrosome. Therefore CD151, α6 and β4 are localized to the different compartments then α3 and β1 integrin subunits. (b,d,e,f,j) Nucleus is visualized by DAPI (blue). (k) Showing the co-localization pattern of CD151 (green) and α6 integrin subunit (red), which is positive in the equatorial segment area. Scale bar represents 1 µm. (l) Co-immunoprecipitation of CD151 with α6 integrin (first lane) and α3 integrin (third lane); asterisk indicates detection of α6 integrin band (∼120 kDa; blue arrow) in the CD151 immunoprecipitate of mouse epididymal sperm lysates. Negative control, immunoprecipitation with rabbit IgG followed by a detection with α6 integrin antibody, is shown in the second lane; other bands probably represent heavy chain of immunoglobulin and non-specific interaction of antibody.

Article Snippet: After electrophoretic separation in 10% polyacrylamide gel and transfer into PVDF membrane, the CD151 immunoprecipate was incubated with mouse antibody against α6 integrin (F-6, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and rabbit antibody α3 integrin (H-43, Santa Cruz Biotechnology, Inc.) diluted 1:100 in PBS overnight at 4 °C.

Techniques: Membrane, Immunoprecipitation, Negative Control

Journal: Scientific reports

Article Title: Expression and distribution of CD151 as a partner of alpha6 integrin in male germ cells.

doi: 10.1038/s41598-020-61334-2

Figure Lengend Snippet: Figure 5. CD151 interactions predicted by docking to the C-terminal domain of the extracellular part of integrin α6. (a) Two predicted binding modes of the α6 C-terminal domain (red) with the CD151 involving its extracellular part. The model shows CD151 in a predicted higher affinity mode (green) as well as in a lower affinity mode (cyan). (b) The predicted positions of CD151 models after a structure superposition to the previously derived models of the α6/β1 (red/blue) and α6/β4 (red/orange) integrin complexes. The model shows that the transmembrane regions of the CD151 are tilted with respect to the membrane plane and the biologically relevant interaction might involve the CD151 and α6 intergrin domain in its more extended conformation.

Article Snippet: After electrophoretic separation in 10% polyacrylamide gel and transfer into PVDF membrane, the CD151 immunoprecipate was incubated with mouse antibody against α6 integrin (F-6, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and rabbit antibody α3 integrin (H-43, Santa Cruz Biotechnology, Inc.) diluted 1:100 in PBS overnight at 4 °C.

Techniques: Binding Assay, Derivative Assay, Membrane