Figure 4 | Leukemia

Figure 4

From: Janus kinase 2 regulates Bcr–Abl signaling in chronic myeloid leukemia

Figure 4

Jak2 inhibition by TG reduced activation of the Ras pathway and PI-3 kinase pathways. (a) Jak2 inhibition of 32Dp210 cells reduced binding of Grb2 to anti-Abl immunoprecipitates. (b) Jak2 inhibition by TG reduced levels of Ras GTP in 32Dp210 cells. These assays were performed with a commercial kit as described by the manufacturer. It involves IP/westerns with their RAS GTP antibody. The kit allows for generation of internal positive and negative controls; the third control is untreated Bcr–Abl+ cells. (c) Quantitation of RAS GTP levels following treatment of 32Dp210 cells with 10 μM TG. The intensity values of the RAS GTP bands were divided by the intensity of the actin band. (d) Jak2 inhibition by TG rapidly reduced levels of pTyr (452 YxxM) Gab2 in 32Dp210 cells. pTyr 452 is believed to bind to regulatory subunit of the PI-3 kinase, which is required for activation of PI-3 kinase. (e) Jak2 inhibition by TG rapidly decreased tyrosine phosphorylation of STAT5 (residue 694). (f) Knockdown of Jak2 reduced pathways related to the Bcr–Abl/Jak2 pathway in CML cell line K562-R. Jak2-specific short hairpin RNA was induced by treatment of cells with doxicycline as in Figure 1. The lysate was western blotted with the antibodies listed.

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