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. 2017 May 15;31(10):1007–1023. doi: 10.1101/gad.297135.117

Figure 2.

Figure 2.

CBL E3 ligases mediate TPO-stimulated JAK2 ubiquitination, degradation and signaling. (A) TF-1/MPL cells were starved and stimulated with or without TPO in the presence of CHX for indicated times. JAK2 and MPL protein levels were determined by Western blot. (B) Quantification of JAK2 and MPL half-lives with or without TPO as shown in A. Error bars denote mean ± SEM. (**) P < 0.01; (***) P < 0.001. (C) JAK2 and MPL half-lives were analyzed in TF-1/hMPL cells with control or double depletion of CBL/CBL-b (Dko + d). (D) JAK2, MPL, and CBL half-lives were analyzed in TF-1/hMPL cells stably expressing empty vector (EV), CBL wild type, or the C381A mutant. (E) TF-1/MPL cells with shLuc or CBL/CBL-b (Dko + d) were treated with or without TPO for 10 min. Lysates were incubated with Flag-K63Ub-TUBE (tandem-repeated Ub-binding entity) and then precipitated with anti-Flag beads. The precipitates were subjected to Western blot with the indicated antibodies. “C” indicates control immunoprecipitation with Flag beads. (F) TF-1/MPL cells stably expressing either EV alone or wild-type or kinase-inactive mutant (Y1007/1008F [YF]) myc-JAK2 were stimulated with or without TPO for 10 min. Cell lysates were pulled down with Flag-K63Ub-TUBE as described in E. Precipitates (top) and total cell lysates (bottom) were subjected to Western blot with the indicated antibodies. The asterisk indicates a possible pTYK2 band that cross-reacts with pJAK2 antibodies. (Endo) Endogenous. (G) TF-1/MPL cells were pretreated with DMSO or 1 µM dasatinib for 1 h and then stimulated with or without TPO for 10 min. Cell lysates were precipitated with anti-pTyr antibody 4G10 followed by anti-CBL blot. Total cell lysates were subjected to Western blot with the indicated antibodies. (H) JAK2 or CBL half-lives were examined in TF-1/MPL cells pretreated with DMSO or 1 µM dasatinib followed by CHX assay. (I,J) TF-1/MPL cells with shLuc or CBL/CBL-b (Dko + d) were stimulated with a graded concentration of TPO for 10 min (I) or with 100 ng/mL TPO for the indicated times (J). Cell lysates were subjected to Western blot with the indicated antibodies.