Western blot

Antibody against IκBα was purchased from the Cell Signaling Technology (#9242); the antibody against α-tubulin from the Santa Cruz Biotechnology (Santa Cruz Biotechnology) and ISG60 has been described before (34). Monocyte-derived macrophages were treated as indicated and whole cell lysates were re-suspended in self-made lysis buffer (20 mmol/L HEPES pH 7.4, 1% Triton-X 100, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 12.5 mmol/L β-glycerophosphate, 2 mmol/L EGTA, 10 mmol/L NaF, 2 mmol/L DTT, 1 mmol/L Na3VO4, 1 mmol/L PMSF plus 1x protease inhibitors). Equal amounts of protein were immunoblotted with IκBα antibody or ISG 60 and a-tubulin antibody. The blots were quantitated by densitometry using the ImageJ software (NIH, Bethesda). The integrated pixel value was determined for each band by multiplying the image intensity by the band area after having subtracted the mean background value.