Fig. 5

Intracellular CYTL1 interrupts MDM2-mediated degradation of NDUFV1 by competitive binding. a The degradation of NDUFV1 over time in the presence of cycloheximide (CHX) was monitored by western blot in MDA-MB-231 cells transfected with HA-CYTL1 or HA-ΔCYTL1 expressing plasmids. b The NDUFV1 protein level in MDA-MB-231 cells before and after treatment of 20 μM MG132 for 6 h. c Co-IP analysis of the interaction between Flag-NDUFV1 and HA-Ub in transfected HEK293T cells. d Co-IP analysis of the interaction between NDUFV1 and HA-Ub in HEK293T cells cotransfected with Myc-NC or Myc-CYTL1 after treatment of 20 μM MG132 for 6 h. e, f NDUFV1 protein expression was determined by western blot in MDA-MB-231 cells transfected with e MDM2 expressing plasmid or f siRNA specific for MDM2. Tubulin was used as a loading control. g Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 in transfected HEK293T cells. h Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 in transfected HEK293T cells cotransfected with HA-NC, HA-CYTL1, or HA-ΔCYTL1. i Schematic representation of NDUFV1 mutants that have truncated sequences. j Co-IP analysis of the interaction between HA-CYTL1 and Flag-NDUFV1 or its three mutants in transfected HEK293T cells. Stars indicate non-specific bands. k Co-IP analysis of the interaction between HA-CYTL1 and Flag-NDUFV1 or its two mutants with deletion of N-terminal sequences. l Co-IP analysis of the interaction between MDM2 and Flag-NDUFV1 or its two mutants. m Cytoplasmic and mitochondrial protein were extracted from HEK293T cells transfected with HA-NC or HA-CYTL1. Co-IP analysis was performed for the interaction between endogenous NDUFV1 and HA-CYTL1. The data are shown as the mean ± SD of three independent experiments. ***P < 0.001