Effect of JAK2 mutations on protein stability and chaperone function for MPL cell-surface expression. (A) Ba/F3-MPL cells expressing either JAK2 WT, V617F, R867Q, or S755R/R938Q and maintained in WEHI-supplemented medium were treated with CHX (50 μg/mL) for 0, 0.5, 1, 2, 5, 8, and 24 hours. Levels of both total JAK2 and MPL proteins were examined by western blotting, and β-actin serves as loading control. Table shows means ± SEM of the half-lives (T1/2) of JAK2 WT and mutants and mature cell-surface MPL interpolated from y = 0.5 on the curves corresponding to half of the protein remaining in CHX-treated cells compared with CHX-untreated cells (n = 3). Significance compared with JAK2 WT T1/2 or cell-surface MPL T1/2 in presence of JAK2 WT was assessed using the 2-tailed Student t test. *P < .05; **P < .01; ***P < .001. (B) Ba/F3 cells expressing the FLAG-tagged MPL and transduced with the bicistronic retroviral pMIGR-IRES-GFP vector encoding either JAK2 WT, V617F, R867Q, S755R/R938Q, S755R, or R938Q were sorted for equal GFP levels and maintained in IL-3–supplemented medium. GFP expression allowed monitoring of JAK2 level in the various cell lines, and MPL cell-surface expression was assessed by flow cytometry using PE fluorescence labeling of the extracellular FLAG tag. Histogram shows the mean fluorescence intensities (MFIs) of PE-labeled cell-surface MPL. (C) Histogram shows means ± SEM of PE MFI in fold related to Ba/F3-MPL cells with no overexpression of JAK2 (Endog.) (n = 3). Significance compared with the Ba/F3–MPL–JAK2 WT condition was calculated using the 2-tailed Student t test. *P < .05.