P mob1

Proteins were isolated by SDS-PAGE and blotted onto a PVDF membrane. Membranes were blocked with 5% milk and incubated with specific primary antibodies, following the same method and incubated with peroxidase-conjugated secondary antibodies. The bands were visualized by an ECL kit (Advansta, Menlo Park, CA, USA). Subsequently, protein grayscale analysis was conducted using Gel-Pro software. The following antibodies were used: ST6Gal-1 (1:1000, Proteintech, 14355–1-AP), p-YAP (Ser127; 1:1000, Cell Signaling Technology, 13008), YAP (1:1000, Cell Signaling Technology, 8418), LATS1 (1:1000, Cell Signaling Technology, 3477), MST1 (1:1000, Cell Signaling Technology, 3682), SAV1 (1:1000, Cell Signaling Technology, 13301), MST2 (1:1000, Cell Signaling Technology, 3952), MOB1 (1:1000, Cell Signaling Technology, 13730), p-MOB1 (1:1000, Cell Signaling Technology, 8699), and GAPDH (1:6000, Bioworld, AP0063).

Western blotting protocol was performed as previously described [20 (link)]. 20–30 µg of protein lysate was used in each experiment. Primary antibodies used for experiments are the following: ERK (Cell Signaling Technology, #4695, 1:1000), p-ERK (Cell Signaling Technology, #4370, 1:1000), AKT (Cell Signaling Technology, #4691, 1:1000), p-AKT (Santa Cruz Biotechnology, sc-7985-R, 1:250), MET (Proteintech, 25869-1-AP, 1:1000), Tubulin (Proteintech, 66031-1-Ig, 1:3000), YAP1 (Cell Signaling Technology, #14074, 1:1000), p-Ser397-YAP1(Cell Signaling Technology, #13619, 1:1000), MOB-1 (Cell Signaling Technology, #13730, 1:1000), p-MOB-1 (Cell Signaling Technology, #8699 S, 1:1000), LATS-1 (Cell Signaling Technology, #3477,1:1000), p-LATS-1 (Cell Signaling Technology, #8654, 1:1000). Anti-mouse (GE Healthcare, United Kingdom, ref. NA931V) or anti-rabbit (GE Healthcare, UK, ref. NA934V) secondary antibodies were used at 1:10000 dilution.

Recombinant human BMP-2 was purchased from DaeWoong Pharmaceutical (Seoul, South Korea). Cell cycle related protein antibodies (cyclin D1, CDK4, p21, p53, p-Smad, and CDK6) and Hippo signaling pathway related protein antibodies (Mst1, Mst2, Sav1, LATS1, p-LATS1, Mob1, p-Mob1, YAP, and p-YAP) were obtained from Cell Signaling Technology (Danvers, MA, USA). The BMP-2 antibody was obtained from Abcam (Cambridge, UK) and BMPR II, RASSF1, Akt, p-Akt, TP63, and β-actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). RASSF1 small interfering RNA (siRNA), YAP siRNA, or control siRNA were purchased from Santa Cruz biotechnology (Dallas, TX, USA). The human TE-8 and TE-12 esophageal cancer cell lines were obtained from Dr. Izzo (Unversity of Texas M.D. Anderson Cancer Center, Houston, TX, USA). DMEM-F12 medium (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (Gibco), 100 mg/ml streptomycin, and 100 IU/ml penicillin (Gibco) was used for cell medium. Cells were maintained under standard conditions at 37 °C in a 5% CO₂ humidified atmosphere.