Rabbit anti human phospho p38

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)].

Western blot analyses were performed as described previously [27 (link)]. In brief, samples of cell lysate (20 μg of protein) were subjected to 12% SDS-PAGE and transferred to PVDF membranes, which were then treated with 5% nonfat milk in 0.1 M phosphate buffer for 1 h at RT to block nonspecific binding of antibody. To measure IL-1β levels, the membranes were then incubated with rabbit anti-human IL-1β (1 : 1000 dilution, GeneTex, Irvine, CA, USA) and then with horseradish peroxidase- (HRP-) conjugated goat anti-rabbit IgG antibodies (1 : 6000 dilution, KPL, Gaithersburg, MA, USA) and bound antibody was detected using Chemiluminescence Reagent Plus. In other studies, the antibodies used were rabbit anti-human phospho-Jun N-treminal kinase (JNK), rabbit anti-human phopho-ERK1/2, rabbit anti-human phospho-p38 (all 1 : 1000 dilution, Cell signaling, Danvers, MA, USA), rabbit anti-human phospho-p65, and rabbit anti-human p65 (all 1 : 1000 dilution, GeneTex) followed by HRP-conjugated goat anti-rabbit IgG antibodies. The intensity of each band was quantified using a densitometer. Antibodies against β-actin, α-tubulin, or GAPDH (all 1 : 1000 dilution, Gene Tex) were used as loading controls.