Horseradish peroxidase labeled secondary goat anti mouse igg antibody

Primary monoclonal antibodies against GRα, GRβ, phospho-p38MAPK, phospho-JNK and β-actin, as well as a horseradish peroxidase-labeled secondary goat anti-mouse IgG antibody, were purchased from Santa Cruz Biotechnology (USA). IL-1β was purchased from PeproTech (England) and RPMI1640 culture medium was purchased from GIBCO (USA). The specific inhibitors of the p38 MAPK pathway (SB203580), ERK pathway (PD98059), PI3K pathway (LY294002), PKC pathway (Ro31-8220) and JNK pathway (SP600125) were all purchased from Calbiochem (USA). [³H]-Dexamethasone was obtained from Amersham (USA). Dexamethasone was purchased from Sigma (USA). The primers and Taqman probes targeting GRα, GRβ, p38 MAPK and JNK were designed and synthesized by Applied Biosystems (USA). All other chemicals used in the study were of analytical grade and obtained from commercial sources.

Frozen colon tissue samples were lysed in RIPA buffer (Qiagen) followed by centrifugation (12,000 rpm, 4°C, 15 minutes), after which the supernatants were stored at −80°C until use. Extracted proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with Tris-buffered saline containing 0.1% Tween 20 (pH 7.6) for 1 hour at room temperature. Subsequently, the PVDF membrane was immunoblotted overnight at 4°C with the primary antibody solution (1 : 1,000). After washing twice with TBST, the membrane was incubated with horseradish peroxidase-labeled secondary goat anti-mouse IgG antibody (Santa Cruz Biotechnology) for 1 hour at room temperature and thereafter washed three times with TBST. The final detection was performed with enhanced chemiluminescence Western blotting reagents (GE Healthcare Bio-Sciences Corp.) and the membranes were exposed to Lumi-Film Chemiluminescent Detection Film (Roche Applied Science).
Loading differences were normalized by using the housekeeping control GAPDH. The primary antibodies used in this study included anti-omentin-1 and anti-GAPDH (both from Santa Cruz Biotechnology).