Anti integrin αvβ3 antibody

Synthetic elastin peptides (VGVAPG, AGVPGLGVG, GRKRK and TAMRA-AGVPGLGVG) were purchased from Proteogenix. Mouse anti-MMP-2 and anti-uPA antibodies were from Calbiochem. Y27632, blebbistatin, U0126, PD150606, lactose, chondroitin sulphate, nifedipine and EDTA were from Sigma-Aldrich. EGCG was purchased from Enzo Life Sciences. Rabbit anti-Hsp90, anti-cleaved caspase-3, anti-integrin αV, anti-p-ERM, mouse anti-αvβ3 and anti-αvβ5 integrin antibodies were from Ozyme. Rabbit anti-RPSA, anti-MMP-14, anti-calpain1, anti-ROCK2, anti-myosin light chain kinase, mouse anti-RPSA, anti-RhoA, anti-ROCK1 and mouse IgM isotype control antibodies were purchased from Abcam. Rabbit anti-p-LIMK-and goat anti-cofilin and anti-actin were from Santa Cruz. Anti-integrin αvβ3 antibody was purchased from Millipore. Annexin-5 alexa fluor^® 568, CellTrace Calcein Red-Orange, AM and DiOC18³ were from Invitrogen.

Cells were harvested with trypsin-EDTA and resuspended in cold PBA (1.0% bovine serum albumin (BSA) and 0.1% sodium azide in PBS), followed by incubation with an anti-integrin αvβ3 antibody (Millipore, Temecula, CA) at 4°C for 60 min. After washing in PBA, cells were resuspended with Alexa Fluor-conjugated second antibodies (Life Technologies, NY); 1:50 in PBA). After an additional 60 min at 4°C, cells were washed and analyzed by flow cytometry (FACSCalibur flow cytometer, Becton Dickinson, NJ), and 10⁴ events were collected and analyzed using WinMDI 2.9 software.