Anti p c raf

Western blot analysis was used to detect proteins in HHGMCs. Cells were lysed with RIPA buffer (BioWorld, Dublin, OH, USA) containing complete protease inhibitors (Roche, Mannheim, Germany) and phosphatase inhibitors (Roche), and the BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA) was used for protein quantification. The protein samples were subsequently electrophoresed on 10% and 15% SDS-PAGE gels and transferred onto PVDF membranes. The membranes were blocked with 5% w/v skim milk/Bovine Serum Albumin (BSA) in TBST (TBS, 0.1% Tween-20) at room temperature for 1 h and incubated overnight at 4°C with the following primary antibodies: anti-HAPLN1, anti-TβRI, anti-TβRII (Abcam, Cambridge, UK), anti-GAPDH, anti-MEK1 (Santa Cruz, Dallas, TX, USA), anti-c-Raf, anti-p-c-Raf, anti-ERK1/2, anti-p-ERK1/2, and anti-p-MEK1/2 (Cell Signaling, Denver, CO, USA). Following washing with TBST, the membranes were incubated with secondary antibody at room temperature for 1 h, washed with TBST and the resulting protein bands were visualized using ECL reagents (GE Healthcare, Buckinghamshire, UK) as a chemiluminescent substrate. The bands were analyzed and quantified using the computer software ImageJ (NIH, Bethesda, MD, USA).