EBNA3C participates within the p62-LC3B complex when proteasome is inhibited.

(A) ~10 x 10⁶ HEK293 cells were transfected with either empty vector (pA3F) or flag-tagged EBNA3C expressing construct. 36h post-transfection cells were either left untreated (DMSO control) or treated with 20 μM MG132 for 4 h. After treatment, cells were harvested and subjected for immunoprecipitation with anti-flag antibody (M2). Precipitated products along with 10% whole cell lysates were run on gel and western blot analysis was performed with indicated antibodies. (B) ~20 x 10⁶ LCLs (LCL#89) either left untreated (DMSO control) or treated with 1 μM MG132 for 12 h, were harvested and subjected for immunoprecipitation using EBNA3C specific mouse monoclonal antibody (A10). Rabbit anti-mouse IgG was used as isotype control. (C) ~5 x 10⁴ HEK293 cells were co-transfected with the indicated expression plasmids using Lipofectamine 3000. 36 h post transfection, cells were further treated with DMSO or 20 μM MG132 for 4 h and subjected for live cell confocal analysis after staining the nucleus with Hochest 33342. (D) For confocal analysis in LCLs, ~5 x 10⁴ LCLs (LCL#1) were treated with DMSO control or 1 μM MG132 for 12 h and subjected for immune staining with EBNA3C, p62, LC3B specific primary antibodies followed by incubation with Alexa Fluor conjugated secondary antibodies. Nuclei were counterstained using DAPI (4’,6’-diamidino-2-phenylindole) before mounting the cells. Each panel in (C-D) corresponds to single experiment of three independent experiments. Scale bars, 5 μm.