The STAT5A/WKR and ▵53C mutants do not inhibit STAT3 activation. Populations of 32D cells expressing either the neomycin resistance gene alone (32D/Neo), STAT5A/WKR (32D/WKR), or STAT5A/▵53C (32D/▵53C) were starved of IL-3 and then stimulated with IL-3 (I), G-CSF (G), or unstimulated (NS). Extracts were precipitated with anti-STAT3 (top 2 panels) or anti-STAT5A (bottom 2 panels) antibodies, blotted to nitrocellulose, and hybridized with anti-phosphotyrosine antibody (top panel of each pair). Membranes were then stripped and reprobed with anti-STAT3 or anti-STAT5A antibodies, respectively (bottom panel of each pair). Expression of STAT5A/▵53C (not recognized by STAT5A antibody) was confirmed by anti-HA Western blot (data not shown).