Phosphotyrosine immunoblot analysis of the IL-3 receptor β subunit and of cellular proteins in TEL-JAK- and TEL-ABL-expressing cells.

(A) The expression of stably transfected TEL-JAK and TEL-ABL fusions in the indicated IL-3-independent Ba/F3 cell lines was evaluated by immunoprecipitation (IP) followed by Western blotting (WB) using a polyclonal rabbit immune serum raised against the amino-terminal part (α N-TEL) of the human TEL protein. Immunoprecipitated complexes are indicated by black arrows; ns, nonspecific. Predicted molecular weights of the chimera are as follows: TEL-Jak1, 75.5 kd; TEL-Jak2, 77.7 kd; TEL-JAK3, 79.3 kd; TEL-ABL, 158.8 kd; murine TEL-Jak2, 50.5 kd; murine TEL-TYK2, 49.4 kd. (B) The IL-3βR subunit was immunoprecipitated from total cell lysates of the indicated IL-3-independent Ba/F3 cell lines and analyzed by immunoblotting with an antiphosphotyrosine antibody (α pTyr). The blot was stripped and reprobed with an anti-IL-3βR antibody. (C) Total cell lysates of the indicated IL-3-independent Ba/F3 cells were subjected to Western blotting using an antiphosphotyrosine antibody. As a control, total cell lysates of parental Ba/F3 cells, starved then stimulated with mIL-3, were loaded. Differences in patterns of cellular protein phosphorylations were reproducibly observed between TEL-JAK- and TEL-ABL-expressing Ba/F3 cells. Note that the weaker pattern of phosphorylation seen in the mTEL-Jak2 line is due to underloading of total cell lysates.