Ab109530

GC cells (AGS, HGC-27, BGC-823, MKN-45, MKN-74, and SGC-7901) and human gastric mucosal epithelial cells (GES-1) were purchased from the Beijing Institute of Cancer Research (Beijing, China). HEK-293T cells were purchased from Procell Biotech (Wuhan, China). GC cells and HEK-293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco Company, USA) containing 10% fetal bovine serum (FBS) 100 unit/mL penicillin and 100 g/mL streptomycin in a humidified atmosphere at 37 °C and 5% CO2. The GES-1 cell was cultured in RPMI-1640 medium (Gibco Company, USA) mixed with 10% FBS. In this experiment, antibodies used as follows, PSMA1(ab109530, Abcam), PSMA1(sc-166073, Santa Cruz), YAP (#4912, CST), YAP (13584-1-AP, Proteintech), TAZ (#83669, CST), TAZ (23306-1-AP, Proteintech), Ki67 (ab1667, Abcam), ACTIN (20536-1-AP, Proteintech), FLAG (20543-1-AP, Proteintech), MYC (#2276, CST), HA (51064-2-AP, Proteintech), PCNA (10205-2-AP, Proteintech), and C-Myc (10828-1-AP, Proteintech).

Splenocytes were lysed in RIPA buffer (Nakalai Tesque) containing protease inhibitors (Roche). The lysates were resolved by SDS-PAGE, and the blots were incubated with anti-β1i, anti-β2, anti-β2i (provided by Shigeo Murata, The University of Tokyo, Tokyo, Japan.), anti-α6 (ab109530), anti-ubiquitin (ab7780, Abcam), anti-β4 (PW8899, Enzo Life Sciences), anti-β5 (PW8895, Enzo Life Sciences), anti-β5i (PTG14859, Proteintech), anti–Ump-1 (15046-1-AP, Proteintech), or anti-actin (A2066, Sigma-Aldrich) antibodies. Then, the blots were incubated with HRP-conjugated goat anti–rabbit IgG antibodies (170–6515; Bio-Rad). The bands were detected with ECL Prime Chemiluminescent Substrate (GE Healthcare) and an ImageQuant LAS-4000 mini system (GE Healthcare). For glycerol gradient analysis, cell lysates were fractionated by linear glycerol density gradient centrifugation (22 hours, 100,000g, 4°C) as described previously (12 (link)).