Constitutive and ligand-activated signaling by Δ5TpoR. Tyrosine phosphorylation of Jak2 and Tyk2 (A) and STAT5 and STAT3 (B) in cells expressing the wtTpoR or the Δ5TpoR was determined in Ba/F3 cells expressing the wtTpoR or Δ5TpoR at equal GFP and protein levels, and after selection with Tpo (for wtTpoR cells) or without any cytokines (for Δ5TpoR). Cells were stimulated or not with 50 ng/mL Tpo for 7 minutes. (A) Cells were lysed and immunoprecipitated with antiphosphotyrosine (P-Y 4G10) antibodies and analyzed by Western blotting with anti-P-Jak2 or anti-P-Tyk2 antibodies. Western blotting using anti-Jak2 or anti-Tyk2 antibodies, respectively, revealed total protein levels of cell lysates used for immunoprecipitations. (B) Western blotting using anti-P-STAT5 or anti-P-STAT3 antibodies revealed levels of STAT5 or STAT3 phosphorylation. Blots were stripped and analyzed for equal protein levels using anti-STAT5 or anti-STAT3 antibodies.