Phospho-amino acid analysis and phosphopeptide map of Stat6.
Ramos cells were starved O/N and metabolically labeled with32P-orthophosphate in phosphate-free medium for 3 hours. Cells were stimulated with IL-4 (100 ng/mL) for 15 minutes and were treated with H7 (200 μmol/L) for 30 minutes before stimulation, if indicated. Cells were lysed in Triton lysis buffer, and Stat6 was immunoprecipitated. Immunocomplexes were separated in 7.5% SDS-PAGE and transferred to PVDF-membrane. (A) Autoradiography of the precipitates. (B) Phospho-amino acid analysis of the excised and hydrolyzed Stat6 bands. (C) For the phosphopeptide map, the Stat6 proteins excised from PVDF-membrane were digested with trypsin. Fragments were separated in the first dimension by electrophoresis at pH 8.9 and in the second dimension by chromatography. Phosphopeptides visualized by autoradiography are shown (numbered 1-8), and origins are marked with a cross.
