Pyap1 s127

RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Hyclone (Logan, VI, USA). The bicinchoninic acid (BCA) protein assay kit, and goat-anti mouse IgM Alexa Flour 488 were purchased from Thermo Scientific (Waltham, MA, USA). The D-Plus^TM CCK cell viability assay kit was purchased from Dongin LS (Seoul, Korea). Mitomycin C was obtained from Sigma (St. Louis, MO, USA). M-MLV reverse transcriptase and RNase inhibitor were purchased from Promega (Madison, WI, USA). dNTP mixture was obtained from TaKaRa Bio (Shiga, Japan). QGreen^TM 2X SybrGreen qPCR Master Mix was purchased from CellSafe (Gyeonggi, Korea). Rabbit poly-clonal antibodies for FAK, p-FAK (Tyr925), Src, p-Src (Tyr416), p-ERK1/2 (Thr202/Tyr204), integrin α5, integrin β1, LATS1, p-LATS1 (Ser909), and p-YAP1 (S127) were purchased from Cell Signaling Technology (Beverly, MA, USA). Rabbit polyclonal antibodies for E-cadherin, β-catenin, ERK1, and CYR61, mouse monoclonal antibodies for YAP1 and α-tubulin, and goat anti-rabbit IgG Texas-Red were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal antibody for G0S2 was obtained from CUSABIO (College Park, MD, USA). All other chemicals and reagents were of highest quality commercially available.

Cells were collected after washes with PBS, lysed in 100 μL of radioimmunoprecipitation (RIPA) buffer containing protease inhibitors (Cat# 04693116001, Roche, Mannheim, Germany) and sonicated. Protein concentrations were determined using the Pierce^TM BCA Protein Assay kit (Cat# 23227, Thermo Fisher Scientific, IL, USA). Primary antibodies against GAPDH (Cat# 2118, RRID: AB_561053, Cell Signaling Technology, MA, USA), YAP1 (Cat# 4912, RRID: AB_2218911, Cell Signaling Technology, MA, USA), p-YAP1 (S127) (Cat# 4911, RRID: AB_2218913, Cell Signaling Technology, MA, USA), CYR61 (Cat# sc-374129, RRID: AB_10947399, Santa Cruz Biotechnology, CA, USA), CCNA2 (Cat# NBP1-31330, RRID: AB_10003781, Novus Biologicals, CO, USA), E2F1 (Cat# A300-766A, RRID: AB_2096774, Bethyl Laboratories, TX, USA), and FOXM1 (Cat# A301-533A, RRID: AB_999586, Bethyl Laboratories, TX, USA) were used.

Immunoblotting was performed as described previously [46 (link), 47 (link)]. The primary antibodies (p-LATS1, LATS1, p-YAP1 (S127), p-YAP1 (S397), YAP1, p-MOB1, MOB1, p-AMPK, AMPK, p27kip1, DNMT1) and peroxidase-conjugated secondary antibody were purchased from Cell Signaling Technology, Inc. The PP1a antibody was purchased from Santa Cruz Biotechnology. The signals were quantified using the G:Box gel imaging system by Syngene (Syngene, Fredric, MD).