anti human integrin β1 ab  (R&D Systems)

Journal: Cells

Article Title: Interaction of Pregnancy-Specific Glycoprotein 1 With Integrin α5β1 Is a Modulator of Extravillous Trophoblast Functions

doi: 10.3390/cells8111369

Figure Lengend Snippet: Co-localization of PSGs with integrin α5, PSG1 expression in EVTs differentiated in vitro, PSG1-mediated adhesion of HTR8/SVneo cells, HIF1α stabilization by CoCl 2 and integrin α5β1 expression in EVT-like cell lines. ( a ) Representative photographs of dual fluorescent immunohistochemistry localizing PSGs (red) and integrin α5 (green) are shown in both individual and dual channels in sections of first-trimester placenta. Scale bars = 45 µm. ctrl = control Ab. ( b ) Immunoblot of cytotrophoblast (CTB) lysates with two different anti-PSG mAbs shows no PSG expression in undifferentiated (0 h) second-trimester CTBs but high expression in the EVTs that were differentiated in vitro (14 h and 20 h) from second-trimester CTBs. ( c ) HTR8/SVneo cells that were pre-incubated with the indicated Abs were seeded on wells coated with 30 μg/mL PSG1-His or control protein. When indicated, cells were pre-treated 4 h with 100 μM CoCl 2 and the treatment was maintained during the experiment. Cell adhesion experiments were carried out as described in . ( d ) Representative image of Western blot showing HIF1α stabilization in HTR8/SVneo and Swan71 cells treated with 100 μM CoCl 2 for 4 h. ( e ) HTR8/SVneo and Swan71 were incubated with anti-integrin α5β1 or isotype control mAb followed by APC-conjugated anti-mouse Ig. MFI = mean fluorescence intensity.

Article Snippet: Following washes, 1 μg/mL of biotin-conjugated anti-human integrin β1 Ab (cat#BAF1778, R&D Systems) was added to the wells for 2 h at room temperature.

Techniques: Expressing, In Vitro, Immunohistochemistry, Control, Western Blot, Incubation, Fluorescence

Journal: Cells

Article Title: Interaction of Pregnancy-Specific Glycoprotein 1 With Integrin α5β1 Is a Modulator of Extravillous Trophoblast Functions

doi: 10.3390/cells8111369

Figure Lengend Snippet: PSG1-mediated adhesion of the EVTs is dependent on a direct interaction with integrin α5β1. Micrographs of Swan71 cells on wells coated with 30 μg/mL PSG1-Fc for 1.5 h in serum-free media ( A , top) or a protein consisting of just the Fc-tag used as control ( B , bottom). Images were taken at 40× magnification. ( B ) HTR8/SVneo and Swan71 cells were seeded in wells coated with PSG1-His or control protein at various concentrations. Cells were incubated for 2 h at 37 °C and the wells were washed to remove non-adherent cells. Cells remaining in the wells were incubated with cell titer aqueous solution and cell adhesion was quantified as described in Materials and Methods. The adhesion of Swan71 cells to wells coated with 60 µg/mL PSG1-His is considered as 100%. ( C ) HTR8/SVneo and Swan71 cells, pre-incubated with 500 μM RGD peptide or control (ctrl) peptide for 30 min at room temperature were seeded in wells coated with 30 μg/mL PSG1-His or control protein. The adhesion of control peptide-treated cells to PSG1-His is considered as 100%. ( D ) HTR8/SVneo cells were pre-incubated with the indicated mAbs for 30 min at RT, after which they were seeded in wells coated with 30 μg/mL PSG1-Fc or control protein. ( E ) HTR8/SVneo cells were pre-incubated with the indicated mAbs for 30 min at RT and seeded on wells coated with 30 μg/mL of native PSG1 (nPSG1) or control protein. ( F ) Swan71 cells and purified primary EVTs, pre-incubated with the indicated mAbs were seeded on wells coated with 30 μg/mL PSG1-His or control protein and adhesion assays were performed in 21% oxygen and CoCl 2 -induced hypoxic-like condition or in 1% oxygen conditions as described in Materials and Methods. The adhesion of control Ab-treated cells to PSG1 is considered as 100% in ( D ), ( E ) and ( F ). ( G ) CHO-K1 (integrin α5-expressing) or CHO-B2 (integrin α5-deficient) cells were seeded on wells coated with 30 μg/mL PSG1-Fc or control protein. The adhesion of CHO-K1 cells to PSG1-Fc is considered as 100%. ( H ) nPSG1 or protein control at 20 μg/mL or bovine fibronectin (FN) at 2 μg/mL was coated on wells. After blocking, 1 μg/mL of α5β1 in 1× TBS/1mM MnCl 2 was added and binding of the integrin was detected with biotin-labeled anti-β1 mAb followed by Streptavidin-HRP. I. Wells were coated with PSG1-Fc or protein control (20 μg/mL). After blocking, 1 μg/mL of α5β1 in 1× TBS/1 mM MnCl 2 was added in combination with 0.5 µM RGD or control peptides and binding of the integrin was detected as indicated in ( H ). The binding of the integrin to the control protein is considered as 1 in ( H ) and ( I ). ( J ) Wells were coated with 20 μg/mL PSG1-Fc or CEACAM9-Fc. After blocking, α5β1 in 1× TBS/1 mM MnCl 2 was added at various concentrations and binding of the integrin was detected as described above. The graph shows values for PSG1 obtained after subtracting the average of the control values. ( K ). HTR8/SVneo cells were seeded on wells coated with the indicated proteins (20 μg/mL) and adhesion experiment was carried out as described in . Cell adhesion to PSG1-His is considered as 100%. Results shown are mean ± S.D. of triplicates from one representative of three independent experiments. p values were obtained by a one-way ANOVA followed by Sidak’s multiple comparison tests for ( H ), ( I ) and ( K ), by a two-way ANOVA followed by Tukey’s multiple comparison tests for ( B ), ( C ), ( E ) and ( F ), or followed by Sidak’s multiple comparison tests for ( G ) (**** p < 0.0001).

Article Snippet: Following washes, 1 μg/mL of biotin-conjugated anti-human integrin β1 Ab (cat#BAF1778, R&D Systems) was added to the wells for 2 h at room temperature.

Techniques: Control, Incubation, Purification, Expressing, Blocking Assay, Binding Assay, Labeling, Comparison