Fig. 5.

HRD1 ubiquitinates CPT2 by targeting K48 ubiquitination. (A) Immunoprecipitation of cell lysates of HRD1 knockdown and shCon cells was performed with CPT2 antibody, and then, lysates were immunoblotted with Ub, HRD1, CPT2, and Vinculin antibody. (B) HEK 293T cells were transfected for 24 h with HA‐Ub, Flag‐CPT2, and Myc‐HRD1 alone or in combination, and treated for 6 h with 10 μm MG132. Immunoprecipitation of cell lysates was performed with Anti‐Flag Affinity Gel, and lysates were immunoblotted with HA, Flag, and Myc antibody. (C) HEK 293T cells were transfected for 24 h with Flag‐CPT2 and Myc‐HRD1 alone or in combination, and treated for 6 h with 10 μm MG132. Immunoprecipitation of cell lysates was performed with Anti‐Flag Affinity Gel, and lysates were immunoblotted with K48‐Ub, Flag, and Myc antibody. (D) HEK 293T cells were transfected for 24 h with HA‐K48Ub, HA‐K48RUb, Flag‐CPT2, and Myc‐HRD1 alone or in combination, and treated for 6 h with 10 μm MG132. Immunoprecipitation of cell lysates was performed with Anti‐Flag Affinity Gel, and lysates were immunoblotted with HA, Flag, and Myc antibody.