Monoclonal anti αsma

The cells were lysed in RIPA Lysis Buffer (Beyotime, China) supplemented with protease inhibitors. The total protein concentration was measured using a BCA protein assay kit (Pierce, MA, USA) according to the manufacturer's instruction. A total of 20 μg protein was separated on 10% SDS-PAGE and transferred onto PVDF membranes (Beyotime). Blocking was performed in 5% nonfat dried milk in Tris-buffered saline containing 0.1% Tween 20 at room temperature for 2 hours. The membranes were incubated overnight at 4°C with primary antibodies, including the following: polyclonal anti-CXCR7 (1 : 400; Abcam, Cambridge, MA, USA), polyclonal anti-pan-AKT (1 : 1000; Abcam), polyclonal anti-phospho-AKT (1 : 500; Abcam), monoclonal anti-ERK1/2 (1 : 5000; Abcam), monoclonal anti-phospho-ERK1/2 (1 : 1000; Abcam), monoclonal anti-β-catenin (1 : 400; Abcam), monoclonal anti-c-Myc (1 : 500; Abcam), polyclonal anti-cyclinD1 (1 : 1000; Abcam), polyclonal anti-survivin (1 : 5000; Abcam), monoclonal anti-α-SMA (1 : 1000; Abcam), and monoclonal anti-GAPDH (1 : 10000; Abcam). The membranes were then washed with TBST three times, incubated with HRP-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) for 2 hours at room temperature, then detected with ECL detection reagents (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as a loading control.