BRISC depletion enhances MPL surface levels as well as MPL-associated membrane proximal JAK2. (A) TF-1/MPL cells with stable depletion of BRCC36 or KIAA0157 were starved and stimulated with or without human TPO in the presence of cycloheximide (CHX) for indicated times. JAK2, pJAK2, and MPL half-lives were determined by WB analysis. Representative blots of 3 independent experiments are shown on the left. Relative protein levels to shRNA-luciferase (shLuc) controls at time 0 for JAK2 and MPL, or to time 0.5 hour for pJAK2, are shown on the right. (B) TF-1/MPL cell lines stably expressing shRNAs to Luc, BRCC36, or KIAA0157 were starved and stimulated with TPO for different times. Quantifications of TPO-stimulated MPL internalization relative to time 0 of the shLuc controls are shown. (C) JAK2 binds MPL upon ligand stimulation. TF1 cells stably expressing shRNAs to Luc or BRCC36 were starved for 3 hours and treated with TPO for indicated times. Cell surface MPL receptors were precipitated with anti-CD110 antibodies in native PBS buffer. Subsequently, cells were extensively washed and permeabilized with NP-40 and subjected to pulldown with Protein G agarose. Precipitates were then blotted with antibodies to pJAK2, JAK2, 4G10 for pMPL, and total MPL. The pulldowns were performed 3 to 5 times, and representative blots are shown. (D) Quantifications of TPO-stimulated pJAK2 and JAK2 associated with cell surface MPL receptor are shown. *P < .05; **P < .01; ***P < .001, 2-tailed Student t test.