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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 8. G-CSF stimulated association of Shp2 with Gab2 requires Gab2 tyrosines 614 and 643.

Figure 8

A. Cells were cotransfected with vector (pcDNA3) or Jak2, and with Flag-tagged- Gab2 (wtGab2), Gab2 Y614F or Gab2 Y643F. Approximately 48 hours after transfection, the cells were treated (+) or not (−) with G-CSF, then lysed. Anti-Flag antibodies were used for immunoprecipitation of Gab2 from aliquots of lysates, and immunoprecipitates were analyzed by sequential Western blotting with antibodies to phosphotyrosine (P-Tyr, top panel), Shp2 (middle panel) and Gab2 (bottom panel). The positions of Gab2 and Shp2 are indicated, and a co-immunoprecipitated ~110kDa phosphoprotein is marked with an arrowhead. G-CSF stimulated the co-immunoprecipitation of Shp2 with Gab2 in the presence of Jak2. Shp2 association with Gab2 was inhibited by mutation of either Y614 or Y643; G-CSF stimulated phosphorylation of Gab2 was strongly inhibited by mutation of Y643.

B. Lysates from the cells transfected with Jak2 and Gab2 constructs were further analyzed by Western blotting using anti-Jak2, to confirm equivalent Jak2 expression in stimulated and unstimulated cells.