Figure 3.

CBL E3 ligases regulate JAK2 stability through the adaptor protein LNK. (A) TF-1/MPL cells expressing either Cas9 alone or Cas9 plus three different gRNAs to LNK were analyzed for depletion efficiency using Western blot. (B) The graph shows the MTT growth assay of the indicated TF-1/hMPL cell lines cultured in TPO. Note that the black and blue lines overlap. (***) P < 0.001, two-tailed Student's t-test. (C) JAK2 and MPL half-lives were determined in TF-1/hMPL cells expressing Cas9 alone or LNK knockout by CHX assay. (D) Cas9 or LNK knockout TF-1/MPL cells were treated with or without TPO for 10 min. Lysates were pulled down with Flag-K63Ub-TUBE and anti-Flag beads. The precipitates were subjected to Western blot with the indicated antibodies. “C” indicates control immunoprecipitation with Flag beads. (E) JAK2 half-lives were determined in TF-1/MPL cells overexpressing EV, LNK wild type, or LNK-R392E by CHX assay. (F) Cas9 control or LNK knockout TF-1/MPL cells were stably infected with retroviruses expressing either EV or CBL wild type. LNK depletion efficiency and CBL expression were analyzed by Western blot. (G) JAK2 and MPL half-lives were determined in the indicated cell lines from F by CHX chase assays. (H) In vitro ubiquitination of JAK2 by CBL was assayed with recombinant JAK2, E1, E2, Ub-biotin, and the CBL TKB + RING domain. The reactions were subjected to Western blot analysis with anti-JAK2 antibodies and streptavidin-HRP. (I) The activity and kinetics of CBL E3 Ub ligase in ubiquitinating JAK2 were assayed using a homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assay.