Rabbit anti human p erk1 2

Western blot analysis was used to detect changes in p-ERK1/2, p-p38, GRα and phospho-serine 226-GR using a rabbit anti-human p-ERK1/2 (1:1,000) antibody (monoclonal antibody; Cell Signaling, Boston, MA, USA; cat. no. 4376S) normalised to total rabbit anti-human ERK1/2 (1:1,000) antibody (monoclonal antibody; Cell Signaling; cat. no. 4695); rabbit anti-human phospho-p38 (1:1,000) antibody (monoclonal antibody; Cell Signaling; cat. no. 4631) normalised to total rabbit anti-human p38 (1:1,000) antibody (monoclonal antibody; Cell Signaling; cat. no. 9212); total mouse anti-human β-actin (1:10,000) antibody (monoclonal antibody; cat. no. A1978; Sigma); or rabbit anti-human polyclonal phospho-GR-Ser226 (1:1,000) antibody (Novus Biologicals, Littleton, CO, USA; cat. no. NB100-92540), normalised to mouse anti-human monoclonal GRα (1;1,000) antibody (BD Biosciences, Franklin Lakes, NJ, USA; cat. no. 611227) as previously outlined in detail [9 (link)].

Serial tissue sections were incubated with either mouse anti-human pAkt1/2/3 (1:100 dilution, Thr308/Thr309/Thr305; Santa Cruz Biotechnology, Dallas, TX, USA) or rabbit anti-human pERK1/2 (1:200 dilution, Thr202/Tyr204; Cell Signaling Technology, Danvers, MA, USA) monoclonal antibody at 4°C overnight, and then incubated with a horseradish peroxidase-conjugated secondary antibody (Maixin-Bio, Fuzhou, China) at room temperature for 45 min. The immunoreactivity was visualized using diaminobenzidine substrate (Maixin-Bio). The positivity was determined independently by three liver pathologists considering both intensity and subcellular localization as reported (9).