Phosopho erk1 2 thr202 tyr204

Western blot was performed as the previously described [26 (link)]. Briefly, total protein was extracted using RIPA buffer added with PMSF and phosphatase inhibitors. Protein concentration was determined using BCA protein assay (Millipore, Switzerland). Then, 20 μg of protein was separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (PVDF, Millipore, USA). After blocked with 5% non-fat milk, the membrane was incubated with primary antibody against PCNA (1:2000), Snail (1:1000), Vimentin (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), integrin β1 (1:1000), Shc (1:1000), phosopho-Shc (1:2000), ERK1/2 (1:1000) or phosopho-ERK1/2 (Thr202/Tyr204) (1:2000) (Cell Signaling Technology, USA), periostin (1:1000), collagen I (1:5000), α-SMA (1:300) or anti-vitamin D receptor antibody (1:1000) (Abcam, USA), GAPDH (1:1000), tubulin (1:1000) or β-actin (1:1000) (Beyotime, China) at 4 °C overnight and the corresponding HRP-conjugated secondary antibody for 1 h at room temperature on the next day. The membrane was developed with enhanced chemiluminescence (ECL; New Cell & Molecular Biotech Co., China).

Western blot was carried out as previously described [26 (link)]. Total proteins were extracted with lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) premixed with phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor (Roche). After samples were loaded into gels, electrophoresis, transferring and immunostaining were conducted. The primary antibodies used were: PCNA (1:2000), vimentin (1:1000), E-cadherin (1:1000), N-cadherin (1:1000), Nanog (1:1000), ERK1/2 (1:1000), phosopho-ERK1/2 (Thr202/Tyr204) (1:2000) (Cell Signaling Technology, USA), Tubulin (1:1000) and GAPDH (1:1000) (Beyotime, China). The immunoreactive bands were detected by enhanced chemiluminescence (New cell & Molecular Biotech, China).