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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 1. Effect of Jak inhibition on G-CSF-stimulated Gab2 tyrosine phosphorylation and Shp2 binding.

Figure 1

A. G-CSF stimulated the phosphorylation of Gab2 and its association with Shp2 in human myeloid cells. AML-193 cells, after serum deprivation for 15 hours, were treated (+) or not (−) with G-CSF (50 ng/ml) for 5 minutes and lysed. Anti-Gab2 immunoprecipitates from lysates were evaluated by SDS-PAGE and Western blotting with anti-phosphotyrosine (top) to identify phosphorylated Gab2 (pGab2). The membranes were then stripped and re-probed with antibodies to Gab2 (middle) and to Shp2 (bottom), to identify total Gab2 protein and co-immunoprecipitated Shp2.

B. AML-193 cells were serum starved for 15 hours, treated (+) or not (−) with AG490 (15 μM), and then stimulated (+) or not (−) with G-CSF (50 ng/ml) for 5 minutes. Anti-Gab2 immunoprecipitates were analyzed by Western blotting with anti-phosphotyrosine to detect phospho-Gab2 (p-Gab2). The membrane was stripped and reprobed with anti-Gab2 to identify total Gab2 (Gab2). Samples of total lysates were analyzed by Western blotting with antibodies to phospho-Jak2 (pJak2) and total Jak2 (Jak2), confirming Jak2 inhibition by AG490. The results indicate that the Jak kinase inhibitor AG490 inhibits G-CSF-stimulated Gab2 phosphorylation.

C. The Jak inhibitor AG490 produced a concentration-dependent reduction in G-CSF-stimulated Gab2 phosphorylation and Shp2 association in human myeloid cells. AML-193 cells were serum starved for 15 hours, treated with the indicated concentration of AG490, and then stimulated (+) or not (−) with 50 ng/ml G-CSF for 5 minutes. Cells were then lysed and half of the lysate used for anti-Gab2 immunoprecipitation. Samples of total lysates were analyzed by Western blotting, as in part B, to confirm Jak2 kinase inhibition (not shown). Anti-Gab2 immunoprecipitates were analyzed by SDS-PAGE and Western blotting with antibodies to phosphotyrosine (top) to identify phosphorylated Gab2 (pGab2), with antibodies to Gab2 (middle) to identify total Gab2 protein, and with antibodies to Shp2 (bottom) to identify co-immunoprecipitated Shp2.