Hrp linked goat anti mouse igg

Dulbecco's modified Eagle medium and fetal bovine serum (Gibco BRL, USA); trypsin, LPS, MTT (Sigma Chemical Co., MO, USA); penicillin and streptomycin (Sunshine Biotechnology, Nanjing, China); and antibodies to YAP, CTGF, Cyr 61, AREG, TEAD1, EP1-EP4, β-catenin, COX-2, MST1, β-catenin siRNA, short hairpin RNA (shRNA) of YAP, COX-2, EP2, MST 1 and HRP-linked goat anti-mouse IgG and horseradish peroxidase (HRP)-linked anti-rabbit IgG were obtained from Santa Cruz (CA, USA). YAP,YAP(5SA), YAP(5SA/S94A) expression plasmids were obtained from Addgene (USA). doxycycline inducible YAP lentivirus expression plasmid (PIN20YAP) was previously described [14] (link). EP1-EP4 antibodies, Butaprost, and AH6809 were from Cayman Chemical (Ann Arbor, MI). Celecoxib, verteporfin, and doxycyclin were purchased from Sigma-Aldrich (St. Louis, MO). Other agents were the highest quality available in market.

Total cellular protein was extracted using a cell lysis buffer. Protein were separated by electrophoresis and transferred to membranes. The membranes were blocked in blocking solution and incubated with mouse monoclonal Ab against AFP, ALB, glucose-6phosphate (G-6P), tryosine-aminotransferase (TAT), α1 antitrypsin (α1AT) and CYP3A4 (1:200; Santa Cruz Biotechnology, Inc., AFP antibody sc-51506, ALB antibody sc-51515, G-6P antibody sc-373886, TAT antibody sc-365512, α1AT antibody sc-73431, CYP3A4 antibody sc-53850) for 1 h at room temperature. After washing, the membranes were incubated for 2 h with horseradish peroxidase (HRP)-linked goat anti-mouse IgG (1:1,000; Santa Cruz Biotechnology, Inc., goat anti-mouse IgG-HRP, sc-2005). The membranes were rinsed for 10 sec in substrate buffer to remove residual detergent. The protein bands were visualized by enhance chemiluminescence and the images were captured in X-ray film. Mouse monoclonal Ab against GAPDH (1:5,000; Santa Cruz Biotechnology, Inc., GAPDH antibody, sc-47724) was used as a housekeeping gene control. The protein quantities were determined relative to the internal optical densities of GAPDH reference standards using ImageJ software.