Epo induces p85 interaction with Cbl. (A) Cbl becomes tyrosine-phosphorylated upon Epo stimulation in γ2A/HA-EpoR cells expressing wild-type JAK2 but not kinase-deficient (KD) JAK2. Cell lysates were subjected to immunoprecipitation by Cbl antibodies and immunoblotted with 4G10 to detect phospho-tyrosine. (B) JAK2 phosphorylates Cbl in vitro. Cbl proteins immunoprecipitated from HEK293T cells were incubated with GST-tagged wild-type or kinase-deficient JAK2 kinase domain in in vitro kinase assay. J2(K), JAK2 kinase domain; J2(KD), kinase-deficient JAK2 kinase domain. (C) Epo induces p85 interaction with Cbl. γ2A/HA-EpoR/JAK2 cells transiently expressing Cbl and T7-tagged p85 were induced with Epo. p85 was immunoprecipitated with T7 antibodies, and the immunoprecipitates were probed with antibodies against Cbl. (D) Epo induces phosphorylation of Y⁷³¹ in the p85-binding motif of Cbl. (E) p85 domain structure. SH3, src-homology 3 domain; RhoGAP, RhoGAP homology domain; SH2, src-homology 2 domain; p110BD, p110 binding domain. (F-H) The C-terminal SH2 domain of p85 mediates inducible binding to EpoR upon stimulation. Biotin-tagged p85 fragments were transiently expressed in γ2A/HA-EpoR/JAK2 cells, isolated with streptavidin agarose, and probed with anti-HA to detect bound EpoR. (G) W622X, Q430X, and D327X are truncations where residues W⁶²², Q⁴³⁰, or D³²⁷ were mutated to a stop codon. IP, immunoprecipitation; IB, immunoblot. *Nonspecific bands.