Stat5 is constitutively tyrosine-phosphorylated in Ba/F3 cells expressing activated TEL-Jak2. Ba/F3 (lanes 1 and 2), Ba/F3 HA-TEL-Jak2 JH1 subclone 18 (lanes 3 and 4), Ba/F3 HA-TEL-Jak2 JH1 subclone 36 (lanes 5 and 6), Ba/F3 HA-TEL-Jak2 JH2 subclone 4 (lanes 7 and 8), Ba/F3 HA-TEL-Jak2 JH2+JH1 subclone 23 (lanes 9 and 10), Ba/F3 HA-TEL-Jak2 JH2+JH1 subclone 31 (lanes 11 and 12), and Ba/F3 HA-TEL-KI-Jak2 subclone 11 (lanes 13 and 14) cells were depleted of cytokine and stimulated in the presence or absence of IL-3. One hundred micrograms of lysate was resolved via SDS-PAGE and transferred to a nitrocellulose membrane. Tyrosine-phosphorylated Stat5 was detected using an activation-specific Stat5 antibody. The membrane was stripped and reprobed with a peptide-specific Stat5 antibody.