Phosphopeptide affinity purification of Stat3 and Stat3β. Biotinylated tyrosine phosphorylated peptide (+P) or nonphosphorylated peptides (−P) were bound to paramagnetic beads and incubated 2 hours at 4°C with whole cell extracts (1.5 mg) prepared from unstimulated DER cells. The peptide-protein complexes were magnetically separated and eluted by boiling in SDS-PAGE sample buffer followed by SDS-PAGE separation. The proteins were transferred to PVDF membrane, developed with Stat3 monoclonal antibody, and visualized by ECL chemiluminescence. The position of prestained molecular-weight markers are indicated on the left and of Stat3 and Stat3β are indicated on the right. The results shown are representative of two experiments.