Rabbit monoclonal anti phospho p38mapk

Following administration with different stimulants, total protein was extracted from cells using radioimmunoprecipitation assay lysis buffer in the presence of PMSF (Beyotime Institute of Biotechnology) and protein concentrations were measured using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). Protein samples were separated using 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Bellerica, MA, USA). Following blocking with 5% non-fat milk or 5% bovine serum albumin, membranes were then incubated with primary antibodies against phosphorylated p38MAPK (rabbit monoclonal anti-phospho-p38MAPK; 1:1,000; Cell Signaling Technologies) and total p38MAPK (rabbit monoclonal anti-p38MAPK; 1:1,000; Cell Signaling Technologies), overnight at 4°C. The membranes were then washed and incubated with horseradish peroxidase-conjugated polyclonal goat anti-rabbit IgG secondary antibodies (1:2,000; Santa Cruz Biotechnology, Inc.) for 1 h. Western blots were developed using an enhanced chemiluminescence detection kit (PerkinElmer, Inc., Waltham, MA, USA) and quantified by densitometry using Gel-Pro Analyzer software 4.0 (Media Cybernetics, Inc.).

Immunoblotting was performed as previously described^{38 (link)–40} with rabbit monoclonal anti phospho-p38MAPK (1:1000, Cell Signaling #4511, Danvers, MA, USA), or mouse monoclonal anti p38MAPK (1:3000, BD Transduction Laboratories #612168, Franklin Lakes, NJ, USA). The signals were detected using chemiluminescence.