Horseradish peroxidase hrp conjugated donkey anti rabbit secondary antibody

BMDMs were generated from either C57BL/6 or mEGFP-RelA knock-in reporter mice. The cells were then treated with different concentrations of cycloheximide (Sigma, 01,810) and harvested at 1, 3, and 6 h post treatment. Cells without cycloheximide treatment served as a control. Whole cell extracts per condition were prepared in ice-cold RIPA cell lysis buffer (Millipore, 20–188). Cell lysates were centrifuged at 13,000 rpm for 20 min at 4°C and equal amounts of protein were resolved with a 4 to 12% Bis-tris Gel/MOPS running buffer system (Invitrogen), and then transferred to nitrocellulose membranes. The membranes were analyzed by Western blotting with the following antibodies: rabbit anti-NF-κB p65 (sc-372), rabbit anti-IκB-α (sc-371), rabbit anti-Rho-GDI (Sigma, R3025) and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit secondary antibody (GE Healthcare, NA934V).

RAW cells, THP1 cells, or hMDMs were lysed in RIPA (Radioimmunoprecipitation assay) buffer (Sigma, R0278) containing a protease inhibitor cocktail (Roche). Cell lysates were quantified by protein assay (Biorad) and equal protein amounts were resolved with a 4 to 12% Bis-tris Gel/MOPS running buffer system (Invitrogen) and transferred to nitrocellulose membranes. The membranes were analyzed by Western blotting with the following antibodies: rabbit anti-IRAK1 (Cell Signaling, 4504S), rabbit anti-IRAK4 (abcam, ab13685 or ab32511), rabbit anti-IRAK2 (abcam, ab62419, and ProSci, 3595), rabbit anti-Rho-GDI (Sigma, R3025), and horseradish peroxidase (HRP)-conjugated donkey anti-rabbit secondary antibody (GE Healthcare).