K48-linked polyubiquitination of Stat5A. (A) A schematic diagram of HA-tagged wild-type (WT) and mutant forms of ubiquitin, in which K to R substitution were introduced at position 48 (K48R) or 63 (K63R). (B) Ubiquitination of Stat5A by wild-type and K63R mutant, but not K48R mutant, ubiquitins. HA-tagged wild-type (HA-Ub-WT), K48R (HA-Ub-K48R), or K63R (HA-Ub-K63R) ubiquitin was cotransfected with Stat5A into COS-7 cells in the presence or absence of JAK2 kinase. Following transfection, Stat5 proteins were pulled down from cell lysates using biotin-labeled double-stranded Stat5-binding oligodeoxynucleotides and avidin-conjugated Sepharose beads. Bound proteins were subjected to Western blot analysis with anti-HA (α-HA) antibody.