Ab108320

hMSCs were fixed with 3.7% formaldehyde for 30 minutes at 4°C and permeabilized with 1% Triton-X for 5 minutes at 37°C. The cells were then stained with primary antibodies against human MyoD (sc-32758, Santa Cruz), Myf5 (sc-302, Santa Cruz, Dallas, TX), Osterix (ab22552, Abcam), CBFA1 (RUNX2) (sc-101145, Santa Cruz), Vinculin (ab129002, Abcam), p130Cas (ab108320, Abcam), SORBS1 (ab4551, Abcam), SORBS3 (GTX-115362, Genetex), Filamin (ab51217, Abcam), or Paxillin (ab32084, Abcam). Corresponding secondary antibodies were conjugated to Alexa Fluor 488 (FITC) or Alexa Fluor 647 (Cy5) (Invitrogen). Nuclei were counterstained with Hoechst dye (Sigma), and the actin cytoskeleton was stained with rhodamine-conjugated phalloidin (Invitrogen). Cells not plated in 96 well plates were imaged with a Nikon Eclipse Ti-S inverted fluorescence microscope equipped with a BD Carv II camera.

Cell lysates were collected by rinsing samples with cold PBS, followed by a five minute lysis in mRIPA buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1% Triton, 1% Na-DOC, 0.1% SDS) with 1 mM EGTA, 1 mM Na3VO4, 10 mM Na4P2O7, and 1 mM PMSF (protease inhibitors). Cell lysates were separated via SDS-PAGE, transferred to PVDF membranes (Bio-Rad), and washed in Buffer A (25 mM Tris-HCl, 150 mM NaCl, 0.1% Tween-20) + 4% SeaBlock (Thermo Fisher Scientific, Waltham, MA) overnight at 4°C. Membranes were incubated with anti-Vinculin (ab18058, Abcam), GAPDH (ab8245, Abcam), ERK2 (ab7948, Abcam), p130Cas (ab108320, Abcam), SORBS1 (ab4551, Abcam), SORBS3 (GTX-115362, Genetex), Filamin (ab51217, Abcam), or Paxillin (ab32084, Abcam) antibodies for 1 hour, washed with Buffer A containing SeaBlock, and incubated in streptavidin horseradish-peroxidase-conjugated secondary antibodies (Bio-Rad) for 30 minutes at room temperature. Immunoblots were visualized using ECL reagent (Pierce). All western blot antibodies were obtained from Abcam (Cambridge, England).