Fig. 3
LPLUNC1 effects on TRIM21-meidated PHB1 ubiquitinating a exogenous PHB1 and TRIM21 proteins form stable complexes. Myc-tagged TRIM21 and Flag-tagged PHB1 were transiently over-expressed in CNE1 cells. At 48 h post-transfection, cells were harvested, and cell lysates were subjected to IP with antibody to Flag or Myc-tag, using normal mouse IgG (IgG) as a control. b, c The PHB1 expression in TRIM21 expression or silencing CNE1 cells was examined by qRT-PCR and western blotting. d Half-life of PHB1 protein is delayed by TRIM21. CNE1 and HNE1 cells were transfected with PCMV-TRIM21 or vector control. At 24 h post transfection, cells were treated with 100 μg/ml cycloheximide (CHX) for indicated time intervals, followed by IB analysis. e TRIM21 mediates poly-ubiquitination of PHB1 ubiquitination. CNE1 cells were transfected with Flag-tagged PHB1 and HA-ubiquitin, plus TRIM21, sh-TRIM21 or their vector controls. At 48 h post transfection, cells were treated with MG132 for 4 h, and then lysates of these cells were subjected to IP with anti-Flag-conjugated beads. Immunoprecipitates were subjected to IB analysis for ubiquitinated PHB1 with antiubiquitin or for indicated proteins. f CNE1 cells were transfected with Flag-tagged PHB1 and HA-ubiquitin, plus TRIM21, sh-TRIM21, LPLUNC1 or their vector controls. At 48 h post transfection, cells were lysed and subjected to IP with anti-Flag-conjugated beads. The immunoprecipitates were subjected to IB analysis for ubiquitinated PHB1 with antiubiquitin or for indicated proteins. g IP analysis using anti Flag antibody followed by IB analysis to detect LPLUNC1 and TRIM21 expression in CNE1 cells. Data shown are representative images or expressed as the mean ± s.d. of each group from three separate experiments. (***P < 0.001 vs. controls)
