N-terminal domain of EBNA3C is important for autophagy mediated degradation in response to proteasomal inhibition.

(A) HEK293 cells transiently transfected with plasmids expressing flag-tagged EBNA3C truncations–residues 1–365, 366–620 and 621–992, either left untreated (DMSO control) or treated with 20 μM MG132 or 50 μM Chloroquine (CQ), were harvested and subjected for western blot analyses with the indicated antibodies. (B) Using a similar experimental set up as described in (A), HEK293 cells transiently transfected with full-length (residues 1–992) and different domains of flag-tagged EBNA3C expression constructs, were subjected to immunoprecipitation (IP) with anti-flag antibody followed by western blot analyses with the indicated antibodies after stripping and reprobing the same membrane. (C) The schematic illustrates known structural motifs and different domains of EBNA3C that are used to determine residues important for autophagy mediated degradation upon proteasomal inhibition. Predicted PEST motifs were derived by using http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind (green: potential; red: poor). (D) HEK293 cells transiently transfected with expression plasmids for flag-tagged EBNA3C N-terminal trunactions (residues 1–365, 50–300, 1–300 and 1–250) either were subjected to western blot analyses with the indicated antibodies after a similar treatment as described in (B). Protein band intensities in (A and D) were quantified by Odyssey imager software and indicated either as bar diagrams or LC3-II/I ratio at the bottom of each corresponding lane. Representative gel pictures are shown of two independent experiments. GAPDH and GFP blots were performed as loading and transfection efficiency controls, respectively.