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. Author manuscript; available in PMC: 2009 Dec 4.
Published in final edited form as: Cell Signal. 2008 Jul 2;20(10):1890–1899. doi: 10.1016/j.cellsig.2008.06.018

Figure 3. Role of G-CSF in the association of Jak2 with Gab2.

Figure 3

A. Human 293 cells were transiently transfected with plasmids encoding Flag-tagged Gab2, the HA-tagged G-CSF receptor, and either vector alone (pcDNA3) or wild-type Jak2 (Jak2). After 48 hours, cells were serum-starved 4 hours, stimulated (+) or not (−) with 50 ng/ml G-CSF and lysed. Equivalent expression of the G-CSF receptor was confirmed by blotting of lysates for the HA epitope tag (not shown). Gab2 was immunoprecipitated from the lysates using anti-Flag antibodies. Immunoprecipitates were analyzed by Western blotting with anti-Gab2 to demonstrate equivalent expression and recovery of Gab2 (middle), and with anti-Jak2 (top), which revealed co-immunoprecipitation of Jak2 with Gab2 that was enhanced by G-CSF treatment. Equivalent expression of Jak2 in the transfected cells +/− G-CSF was confirmed by blotting whole cell lysates with anti-Jak2 (bottom panel).

B. Serum starved DT40GR cells were treated (+) or not (−) with 50 ng/ml G-CSF for the times indicated, then lysed. Anti-Gab2 immunoprecipitates from cell lysates were evaluated by Western blotting after SDS-PAGE. Equivalent recovery and sample loading was confirmed using anti-Gab2 (bottom panel). Antibodies to Jak2 and phosphorylated Jak2 (middle and upper panels, respectively) revealed co-immunoprecipitation of Jak2 with Gab2. The association between Jak2 with Gab2, minimal under basal conditions, transiently increased after addition of G-CSF.