Fig. 6: TRIM2 facilitates K48-linked polyubiquitination of BNIP3.

A Flag-BNIP3 and HA-Ub plasmids were co-transfected into HEK293T cells in the presence or absence of TRIM2. Polyubiquitination of BNIP3 was quantified by co-immunoprecipitation and western blot analysis. B The indicated plasmids were co-transfected into HEK293T cells for 24 hours. The absence and level of ubiquitination of BNIP3 were then detected by co-immunoprecipitation with anti-Flag tag antibodies and immunoblotting (IB) with the indicated antibodies. C Flag-BNIP3 and HA-Ub plasmids were co-transfected into HEK293T cells for 24 hours, with or without TRIM2. Polyubiquitination of BNIP3 was detected by IB with a specific antibody for Ub, K48 Ub, or K63 Ub after immunoprecipitation. D HEK293T cells were co-transfected with the indicated plasmids for 24 hours. The absence and level of ubiquitination of BNIP3 were then detected by co-immunoprecipitation with anti-Flag tag antibodies, and IB with the indicated antibodies. E The endogenous ubiquitination of BNIP3 in Caco-2 cells transfected with HA-TRIM2 or HA-Vector plasmids was detected by IB with the specific antibody for Ub, K48, or K63 after immunoprecipitation. F The endogenous BNIP3 ubiquitination in Caco-2 cells infected with Lenti-si-TRIM2 or Lenti-si-Control virus was detected by IB with the specific antibody for Ub, K48, or K63 after immunoprecipitation. G A graphical representation of the 12-point mutation site in BNIP3, in which all lysine residues (K) were replaced with arginine (R). H HEK293T cells were co-transfected with the indicated plasmids for 24 hours. The polyubiquitination sites of BNIP3 by TRIM2 were identified in the lysates of HEK293T cells by IB with an anti-HA antibody after immunoprecipitation with anti-Flag tag antibodies.