Rabbit anti ikb

Cells were harvested and lysed in NP-40 lysis buffer (Fluka, USA) with protease and phosphatase inhibitors (ThermoFisher). Protein concentrations were measured using the Bradford Protein Assay Kit (Bio-Rad, USA). Equal amounts of protein were separated using 12% SDS-PAGE and transferred to PVDF membranes (Bio-Rad). Afterward, the membranes were blocked with 5% skim milk, incubated overnight at 4°C with each primary antibody, and then washed three times with TBST (25 mM Tris buffer, 0.15 M NaCl, 0.05% Tween 20). The membrane was incubated for 3 h at room temperature with the corresponding horseradish peroxidase (HRP)- conjugated secondary antibody and rewashed three times with TBST. Proteins were visualized using the ECL detection kit (Invitrogen). The antibodies used are as follows: Primary antibodies: rabbit anti-COX-2; rabbit anti- iNOS; rabbit anti-phospho and total-p44/p42 MAPK (Erk1/2); rabbit anti-phospho and total-p38 MAPK; rabbit anti-phospho and total JNK; rabbit anti-IkB (all antibodies mentioned above were from Cell Signaling Technology, USA). Secondary antibodies: mouse anti-β-actin (Sigma-Aldrich, USA); and HRP-conjugated secondary antibody (Promega).