Ab59337

For Western blot analyses, cell extractions were cleared by 13300 × g for 15 min at 4°C. Protein concentration was determined by BCA Protein Assay Kit (APPLYGEN, Beijing, China). The protein samples were separated by 8% SDS-PAGE and transferred to a nitrocellulose membrane using a Trans-blot unit (Bio-Rad Laboratories) for 1.5 hour at 250 mA. The membranes were blocked with 5% (wt/vol) skim milk and 0.1% (vol/vol) Tween-20 in TBS (pH 7.4) for 1 hour at room temperature (25°C). Primary antibodies against BMP4 (1 : 500, ab39973, Abcam, UK), p38 MAPK (1 : 200, sc-7149, Santa Cruz Biotechnology, USA), phospho-p38 (p-p38) MAPK (1 : 1,000, 9216, Cell Signaling Technology, USA), Sgk1 (1 : 500, ab59337, Abcam, UK), p-Sgk1 (1 : 1,000, 44-1260G, ThermoFisher, USA), Nedd4-2 (1 : 500, 4013, Cell Signaling Technology, USA), p-Nedd4-2 (1 : 500, ab168349, Abcam, UK), and GAPDH (1 : 5,000, ab8245, Abcam, UK) were incubated with the membranes overnight at 4°C. After washing with TBS-T, the membranes were incubated for 1 hour at room temperature with the corresponding secondary antibodies (1 : 10,000). All membranes were washed with TBS-T, and the bands were quantified by using the Odyssey infrared imaging system (LI-COR) and Odyssey v3.0 software.

GSK650394, 3-methyladenine (3MA) and Rapamycin were purchased from Selleck. Acridine orange (AO) was obtained from Beyotime (Haimen, China). Antibodies against E-cadherin (#14472), N-cadherin (#13116), Vimentin (#5741), LC3B (#3868), and GAPDH (#5174) were purchased from Cell Signaling Technology (Beverl y, MA, USA). Antibodies against SGK1 (ab59337) and P62 (ab56416) were purchased from Abcam (Cambridge, USA). Antibodies against MMP3 (sc-6839) and MMP9 (sc-21,733) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies (HRP-conjugated sheep anti-rabbit antibodies or HRP-conjugated sheep anti-mouse antibodies) for western blot were obtained from Millipore (Shanghai, China).