Figure 3
From: Janus kinase 2 regulates Bcr–Abl signaling in chronic myeloid leukemia

Jak2 inhibition by TG101209 induced dephosphorylation of Bcr–Abl Tyr177 and reduction of Bcr–Abl. (a) Bcr–Abl pTyr177 and Bcr–Abl levels were reduced in 32Dp210 cells treated with indicated doses of Jak2 inhibitor TG for 16 h. Anti-Abl immunoprecipitates (IP) (P6D monoclonal antibody, custom made) were probed by western blotting (WB) with sequence-specific Bcr pTyr177 antibody (Novus Biologicals, Littleton, CO, USA) and anti-Abl 8E9. Anti-actin (Sigma Chem Co., St Louis, MO, USA) blots were performed on the supernatant before IP as a loading control. IQ represents normalized intensity performed by estimating the band intensity and dividing that by the intensity of the actin band. (b) Jak2 inhibition by TG rapidly reduced pTyr177 Bcr–Abl but had little effect on the level of Bcr–Abl levels. 32Dp210 cells were treated with the 10 μM TG for various times up to 2 h and assayed by western blotting. (c) Jak2 inhibition by TG in CML cell line K562-R reduced levels of Bcr–Abl pTyr177 and Bcr–Abl protein. (d) Cells from a blast crisis (78% blasts) CML patient showed reduced levels of pTyr177 Bcr–Abl and Bcr–Abl by Jak2 inhibition. Monocytes were isolated by Histopaque (Sigma Chem Co.) separation and grown for 24 h in culture medium without growth factors before TG treatment. (e) Jak2 inhibition by TG rapidly reduced levels of pTyr-containing proteins. Lysates were blotted with 4G10 anti-pTyr antibody. (f) CD34+ progenitor cells from a blast crisis CML patient (98% blasts) showed rapid reduction of pTyr177 Bcr–Abl and Bcr–Abl upon treatment with TG for up to 2 h. After isolation of monocytes as in panel d, cells were then fractionated on CD34 antibody magnetic beads. An aliquot assayed by flow cytometry showed that the sample was 87%+ for CD34+ cells. The CD34+ cells were treated with 10 μM TG for up to 2 h in cell culture. (g) Jak2 inhibition by TG reduced levels of pTyr177 Bcr–Abl, Bcr–Abl, Jak2, pTyr1007 Jak2 in human cord blood CD34+ progenitor cells transduced with BCR–ABL as described.12 TG treatment was done for 6 h. (h) Treatment of 32Dp210 cells with IM had little effect on levels of pTyr177 Bcr–Abl and Bcr–Abl protein levels over a 3-h period.