P lats1 ser909

Cells were washed in PBS, dissolved in 60‐100 μL solubilizing solution (9M urea, 2% NP40), and subjected to immunoblotting as previously described.³ (link) Primary Abs recognizing the following proteins were used: YAP1 (D8H1X), TAZ (v386), phospho‐(p)‐YAP1 (Ser127), p‐YAP1 (Ser397), p‐TAZ (S89), LATS1, and p‐LATS1 (Ser909) (all from Cell Signaling Technology), actin (Sigma), or GAPDH (Wako Chemical). Horseradish peroxidase‐conjugated anti‐rabbit Ab (Cell Signaling Technology) was used as the secondary Ab. Band intensities were determined by Fujifilm Multi Gauge software or the ImageJ program. The YAP1 and TAZ protein levels were normalized to actin or GAPDH, as indicated. Phosphorylated YAP1, p‐TAZ, and p‐LATS1 were normalized to total YAP1, TAZ, and LATS1 levels, respectively.

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.