Gab1 and Gab2 are phosphorylated on tyrosine in response to various stimuli. (A) Cytokines and growth factor stimuli. Various cell lines were stimulated by the indicated cytokines or growth factors. Anti-Gab1 and anti-Gab2 immunoprecipitates were analyzed by immunoblotting with anti-phosphotyrosine, anti-Gab1, and anti-Gab2 antibodies. Note that Gab2 was tyrosine-phosphorylated in unstimulated MO7E cells, but the band of Gab2 was shifted upon SCF stimulation, indicating that Gab2 was hyper-phosphorylated in the stimulated cells. HepG2, KT-3, TF-1, NIH3T3, and MO7E were human hepatoblastoma, human (Lennert’s) T lymphoma, human erythroleukemia, mouse fibroblast, and human megakaryocytic leukemia cell lines, respectively. HepG2, TF-1, and MO7E cells express both Gab1 and Gab2. But KT-3 and NIH3T3 cells do not express Gab1 or Gab2, respectively (confirmed by RT-PCR, data not shown). Proteins of 115 to 120 kD detected in the Gab2 immunoprecipitates from HepG2, NIH3T3, and MO7E cells are likely Gab1 proteins cross-reacted with anti-Gab2 antibodies. (B) TCR and (C) BCR stimuli. Jurkat cells were stimulated with anti-CD3 antibody or left unstimulated. Ramos cells were stimulated with anti-IgM antibody or left unstimulated. Cell lysates were immunoprecipitated with anti-Gab1 or anti-Gab2 antibodies and subjected to immunoblotting with anti-phosphotyrosine, anti-Gab1, anti-Gab2, anti-SHP2, and anti-p85 antibodies. Expression of Gab2 in Ramos cells was not detected by immunoblotting and RT-PCR (data not shown).