Figure 5 | Leukemia

Figure 5

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

Figure 5

A new Jak2 inhibitor WP1193 rapidly reduced levels of pTyr177 Bcr–Abl, Bcr–Abl protein and levels of pTyr Bcr–Abl in 32Dp210 cells. (a) Structure of WP1193 and AG490. AG490 is a known Jak kinase inhibitor. (b) Jak2 inhibitor WP1193 inhibits the ability of recombinant Jak2 (JH1-JH2) to phosphorylate Tyr177 Bcr peptide. The same methods were used as described in Figure 2. (c) Top panel: Jak2 inhibitor WP1193 rapidly inhibited phosphorylation of Tyr177 of Bcr–Abl and reduced levels of Bcr–Abl in 32Dp210 cells. 32Dp210 cells were lysed and immunoprecipitated with anti-Abl p6D monoclonal antibody as described in Figure 2a. Bottom panel: 32Dp210 cells were treated for up to 1 h with 10 μM WP1193, lysates were made and immunoprecipitated with anti-Jak2 antibody. Western blots were probed with anti-pTyr 4G10. (d) Jak2 inhibition by WP1193 causes rapid reduction of pTyr177 Bcr–Abl and the Bcr–Abl protein in BaF3 cells expressing IM-resistant form of Bcr–Abl containing the T315I mutation. (e) Jak2 inhibition by WP1193 caused rapid reduction of pTyr177 Bcr–Abl in cells from a blast crisis CML patient. Monocytes from patients were isolated by Histopaque (Sigma Chem Co.,) separation and grown for 24 h in culture medium without growth factors before WP1193 treatment. (f) The level of the Y177F Bcr–Abl mutant is decreased in amount similar to wild-type Bcr–Abl as a result of Jak2 inhibition. 32D cells were transfected with either Y177F BCR–ABL mutant or wild-type BCR–ABL. Cells were allowed to grow in the absence of IL-3 for about 3 weeks. Cells were treated with 10 μM WP1193 for 30 min. Lysates were analyzed by western blotting with anti-Abl 8E9, anti-Tyr 177 Bcr, anti-Jak2 pTyr 1007/8 and anti-Jak2 antibodies. (g) Levels of Grb2 were rapidly reduced in anti-Abl immune complexes containing Bcr–Abl. (h) Levels of Ras GTP were rapidly reduced in 32Dp210 cells treated with Jak2 inhibitor WP1193. Experiments were performed as in Figure 4b. (i) Quantitation of RasGTP levels in 32Dp210 cells treated with Jak2 inhibitor WP1193. Quantitation was done as shown in Figure 4c.

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