The STAT5A mutants are tyrosine-phosphorylated by JAK2, but the STAT5A/WKR mutant fails to heterodimerize with STAT5B. Wild-type or mutant STAT5A was coexpressed in 293T cells with STAT5B with or without JAK2; in the left two lanes, STAT5B alone was expressed with and without JAK2. Proteins were immunoprecipitated with anti-HA (-HA) or anti-STAT5B (-5B) antibodies and blotted with anti-HA (top panel), anti-phosphotyrosine (middle panel), or anti-STAT5B (bottom panel) antibodies. Note that the anti-STAT5B antibody also recognizes STAT5A and the STAT5A mutants with the exception of ▵53C, which is lacking the C-terminal epitope recognized by this antisera. The positions of the STAT5A and STAT5B proteins are indicated by the arrowheads at right. The STAT5A point mutants (5A-PM) migrate more slowly than STAT5B due to additional amino acids at the C-terminus and the HA epitope tag at the N-terminus. When coexpressed with JAK2, STAT5B migrates as three distinct forms (left 2 lanes, bottom panel), of which the two most slowly migrating forms are tyrosine phosphorylated (5B-PY, middle panel).