FIGURE 2.

Alcohol alters IRAK-M and IRAK-1 levels without any effect on CD14 and TLR4 expression. A, Human monocytes were either exposed to 25 mM alcohol (Et) for 1 day (acute) or 7 days (chronic), stained with CD14 and TLR4 Abs, and analyzed by flow cytometry. Iso-type IgG Abs were used as negative controls. B, Human monocytes were exposed to 25 mM alcohol for 7 days (Chr) followed by LPS (100 ng/ml) for 15 min. Immunoprecipitation (IP) of the cytoplasmic extracts with IRAK-1 Ab in lysis buffer was conducted as described in Materials and Methods. The kinase assay was performed with MBP (myelin basic protein) as substrate. The proteins were separated on SDS-PAGE and the gel depicts the ³²P-phosphorylated MBP. Equal protein in the IP samples was determined by immunoblotting (WB) with IRAK-1 Abs. The bar graph below represents the cpm incorporated in the substrate as measured by scintillation counter. (compared with unst; *, p < 0.01; compared with LPS; **, p < 0.05; n = 4). C, Human monocytes were exposed to 25 mM alcohol (Et) for 1 day (Ac) or 7 days (Chr) followed by LPS (100 ng/ml) treatment for 6 h and IRAK-M mRNA determination by real-time PCR using specific IRAK-M primers as described in Materials and Methods. The bar graph represents fold induction of mRNA ± SE (compared with unst; *, p < 0.01; compared with LPS; **, p < 0.02; n = 4). D, Monocytes were exposed to 25 mM alcohol (Et) for 1 day (Ac) or 7 days (Chr) followed by LPS (100 ng/ml) treatment for 24 h and IRAK-M protein levels determined by immunoblotting using anti-IRAK-M Abs as described in Materials and Methods. Equal loading of protein is demonstrated using an internal control anti-β-actin Ab.