CD11b is phosphorylated on Ser1126 in neutrophils. (A) COS cells were transfected with wt Mac-1 or left untransfected. Cell lysates were analyzed by Western blotting with the pCD11b antibody. (B) Western blot of CD11b/CD18 integrin immunoprecipitated from lysed peripheral blood neutrophils detected with pAb P-CD11b. NB indicates no blocking peptide present; BP, blocking peptide present, PP, CD11b phosphopeptide present. (C) Fluorescence-activated cell sorting (FACS) analysis of transfectants shows that Mac-1 heterodimers are not expressed on untransfected Jβ2.7 cells (2 top panels). The expression level of wt Mac-1 is comparable to that of the S1126A–Mac-1 transfectants (2 bottom panels). MEM170, CD11b-antibody; IB4, CD18-antibody. (D) Wt and S1126A-CD11b–transfected Jβ2.7 cells were pretreated with 1.5 μM okadaic acid (OA) and activated with 200 nM PDBu for 30 minutes or left untreated (C). Lysates from cells were analyzed by Western blotting with the pCD11b antibody (top). CD18 is shown as a loading control (bottom). Dotted lines represent the background fluorescence.