Mouse anti β1 integrin

Cells were fixed using 4% paraformaldehyde/PBS for 15 min at room temperature, washed three times with washing buffer (0.1% Tween-20/PBS, 5 min), and permeabilized with 1% Triton X-100/PBS for 30 min. Samples were incubated in blocking buffer (3% BSA, 2% goat serum in PBS) for 30 min, then incubated with various primary antibodies including mouse anti-YAP (Santa Cruz Biotechnology, sc-101199), mouse anti-αVβ3-integrin (Abcam ab78614), mouse anti-β1-integrin (Abcam ab24693), rabbit anti-paxillin (Abcam ab32084), rabbit anti-pFAK (Abcam ab81298), mouse anti-RUNX2 (Abcam ab76956) overnight at 4 °C on a shaker. All antibodies were diluted in buffer at 1:100 except YAP antibody was diluted at 1:300. After washing, samples were incubated with corresponding secondary antibodies including Alexa 488 Goat-anti-mouse (Invitrogen A11001), rhodamine goat-anti-rabbit (Millipore AP132), rhodamine-phalloidin (Sigma P1951) for 1 h at room temperature on a shaker. All secondary antibodies were diluted at 1:300. Cell nucleus counter stain was performed using Hoeschst nuclear stain (Cell Signaling Technology 4082S, 2 ug/mL). Samples were washed with washing buffer (three times, 5 min per wash) before being imaged using a confocal microscope (40x oil immersion, Leica SP8 confocal system). All images were processed using open-source Fiji software [35 (link), 36 (link)].