Fig. 5. HUWE1 ubiquitinates and degrades TfR1.

A Co-immunoprecipitation of HA-TfR1 with Flag-HUWE1 in HEK293T cells. Exogenous HUWE1 was immunoprecipitated using Flag-M2 beads, and the immunoprecipitants were immunoblotted with HA antibody. B Co-immunoprecipitation of Huwe1 with TfR1 in mouse liver hepatoma Hepa-1c1c7 cells. Endogenous TfR1 was immunoprecipitated using anti-TfR1, and the immunoprecipitants were immunoblotted with Huwe1 antibody. IgG, immunoglobulin G. C 293T cells were transfected with increasing levels of Flag-HUWE1, endogenous TfR1 was detected by western blot. Huwe1 WT and KO MEFs were treated with 15 μg/ml cycloheximide (CHX) (D), 10 µM MG132 or 10 µM Chloroquine (CQ) (E), endogenous TfR1 was immunoblotted. Mcl-1 was used as a positive control. F Immunoblotting of Huwe1 and TfR1 in hepatocytes isolated from Huwe1 WT and KO mice. G Hepatic Huwe1 and TfR1 were immunoblotted in Huwe1 WT and HKO mice challenged by CCl₄ for 12 h. (H) Hepatic TfR1 were detected by immunoblotting in Huwe1 WT and HKO mice subjected to I/R for 6 h. TfR1 protein levels were normalized to those of Gapdh. *p < 0.05, compared in the indicated groups. I Ubiquitination of HA-TfR1 in HEK293T cells co-expressed with empty vector or Flag-HUWE1 treated by MG132 for 4 h before harvesting. J Recombinant TfR1 (100 nM) was incubated with ATP regenerating buffer, E1, E2, recombinant proteins of His tagged HECT domain of HUWE1 (10 nM, or 40 nM), and ubiquitin in a 30 μl reaction volume for 1 h at 37 °C. Three independent experiments were performed to confirm the ubiquitination of TfR1 by HUWE1 in vitro. K Immunoprecipitation analysis from HEK293T cells transiently co-transfected with HA-TfR1, Flag-HUWE1 and Wildtype or K48-Ubiquitin. The TfR1 bands in C–H were quantified with the Image J software, and the ratio of TfR1 to Tubulin or Gapdh in each lane was indicated on the bottom.