BRISC depletion enhances JAK2 K63-ubiquitination. (A) K63-ubiquitination of JAK2 correlates with its activity. BaF3/hMpl cells were stimulated with TPO for indicated times. Left panel: Lysates were incubated with Flag-K63TUBE followed by IP with anti-Flag beads. The blots were probed with indicated antibodies. Right panel: Lysates were precipitated with anti-JAK2 serum, and the blot was probed with TUBE1-biotin followed by detection with streptavidin-conjugated horseradish peroxidase secondary antibody for Far-Western detection. The blot was then stripped and probed with indicated antibodies. (B-D) BRISC depletion increases JAK2 K63Ub. (B) TF-1/MPL cells expressing shRNA-mediated knockdown to either controls Luciferase (Luc) or Brcc36 (B36) were pretreated with or without Ubc13 inhibitors and stimulated with TPO for 0 or 10 min. Flag-K63TUBE pulldown was performed as in panel A and blotted with indicated antibodies. (C) TF-1/MPL cells expressing either shRNA to Luc or B36 were pretreated with or without MG132 and stimulated with TPO for 0 or 20 min. Cell lysates were pulled down with Flag-K48TUBE followed by WB analysis with indicated antibodies. (D-E) K63Ub-TUBE pulldowns were performed similarly in TF-1/MPL cells expressing either shRNA to Luc, B36, or KIAA (D) or 32D/Mpl cells expressing either shRNA to Luc or M40 (E). WB analysis of total cell lysates is indicated at “Input” in the right panels. C, control immunoglobulin G; Mono. Ab., monoclonal antibody.