Peroxidase linked igg

For protein extraction, whole cells were lysed in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 30 mM NaF, 1 mM Na₃VO₄, 0.1 mM PMSF, protease inhibitors, and 1% Nonidet-P40). The supernatant was harvested and concentrated using a Vivaspin Turbo 15 (Sartorius Stedim, Gottingen, Germany). For normalization, the same number of cells was plated after CG loading or without CG loading for control, and the GAPDH expression of each condition was investigated using western blot analysis. Protein lysates (30 μg) and concentrated supernatants were resolved by 10–12% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Piscataway, NJ, USA), and then probed with primary antibodies against SOX9 (EMD Millipore, Billerica, MA, USA), BMP4 (Santa Cruz Biotechnology, Inc., Dallas, Texas USA), and GAPDH (Abcam). After extensive washing, primary antibodies were detected by peroxidase-linked IgG (1:5000; Abcam) and visualized using an ECL kit (WESTSAVE GOLD, AbFrontier, Geumcheon-gu, Seoul, South Korea). The intensity of western blot bands was quantified using ImageJ (1.40).

Cellular protein was harvested by using RIPA buffer (Sigma) and quantified by using a BCA protein assay (GenDEPOT). Equal protein amounts were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Amersham Pharmacia Biotech). The membranes were incubated overnight with specific antibodies against α-SMA (Abcam), pSMAD2 (Thermofisher), SMAD2 (Thermofisher) and anti-GAPDH (Abcam). The next day, the membranes were washed, incubated with a peroxidase-linked IgG (Abcam), and visualized by using an ECL kit (WESTSAVE Gold).