Western Blot Analysis of Cell Signaling Proteins

Western blot assay was performed as described previously [21 (link)]. Tissues and cell lysates were prepared by using RIPA Reagent (Beyotime, Shanghai, China) and the concentration was measured by BCA kit (Thermo Fisher Scientific). Proteins were loaded on 10% SDS-PAGE and were transferred onto PVDF membranes (Thermo Fisher Scientific). The membranes were immersed in 5% skim milk powder for 2 h, and then incubated at 4 °C overnight with anti-CyclinD1 (1:200, Abcam, Cambridge, MA, USA), anti-CDK4 (1:2000, Abcam), anti-PARP (1:1000, Cell Signaling Technology, Shanghai, China), anti-Cleaved PARP (1:1000, Abcam), anti-Cleaved caspase-3 (1:500, Abcam), anti-E-cadherin (1:1000, Cell Signaling Technology), anti-N-cadherin (1 µg/mL, Abcam), anti-Vimentin (1:1000, Abcam), anti-DLX2 (1:500, Abcam), Ki67 (1:1000, Abcam) or anti-GAPDH (1:2500, Abcam). Next, membranes were washed and probed with the HRP-labeled secondary antibody (Abcam) for 1 h at 37 °C. Membranes were visualized via the ECL regent (Amersham Biosciences, Buckinghamshire, UK).