⁵⁸⁵Ser in β_c is phosphorylated in vivo by GM-CSF. (A) The anti–phospho-⁵⁸⁵Serβ_c antibody specifically recognizes the phosphorylated CLGPPHSRSLDILG peptide. Dot blots were prepared on nitrocellulose filters of either the nonphosphorylated or the serine-phosphorylated CLGPPHSRSLPDILG peptide and the scrambled peptide CLPLSGPDSHIRGPL before probing with anti–phospho-⁵⁸⁵Serβ_c antibody. (B) The serine-phosphorylated CLGPPHSRSLPDILG peptide specifically inhibits the binding of anti–phospho-⁵⁸⁵Serβ_c antibody to β_c. Lysates of HEK293T cells transfected with wild-type β_c were immunoprecipitated with anti-β_cantibody (MoAb 8E4). Immunoprecipitates were run on 7.5% SDS-PAGE under reducing conditions. Anti–phospho-⁵⁸⁵Serβ_c antibody was then preincubated with either medium (none), 100-fold molar excess of the serine-phosphorylated (1) or nonphosphorylated (2) CLGPPHSRSLPDILG peptide, or the scrambled peptide CLPLSGPDSHIRGPL (3). The filters were then Western blotted and probed with the pretreated anti–phospho-⁵⁸⁵Serβ_c antibody. (C) Upregulation of ⁵⁸⁵Ser phosphorylation by GM-CSF. M1 cells expressing GM-CSFR and β_c were either untreated (−) or stimulated with GM-CSF (2 ng/mL) for 30 seconds (+). Lysates of the M1 cells were immunoprecipitated with anti-β_cantibody (MoAb 8E4) and the immunoprecipitates were run on 10% SDS-PAGE under reducing conditions. The filters were then Western blotted with either anti–phospho-⁵⁸⁵Serβ_cantibody or the anti-β_c antibody (MoAb 1C1).