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EBNA3C N-terminal domain is degraded in both nuclear and cytoplsamic fractions upon proteasomal inhibition.

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posted on 2020-02-24, 18:52 authored by Chandrima Gain, Samaresh Malik, Shaoni Bhattacharjee, Arijit Ghosh, Erle S. Robertson, Benu Brata Das, Abhik Saha

(A) Schematic shows the predicted nuclear export signal (NES) sequences of EBNA3C, derived from http://www.cbs.dtu.dk/services/NetNES/. (B-C) HEK293 cells were transiently transfected with the indicated expression plasmids. 36 h post-transfection cells were further treated with DMSO control or 20 μM MG132 for 4 h and subjected to (B) subcellular fractionation followed by western blot analyses using the indicated antibodies and (C) live cell confocal analyses. (D-E) LCLs were either left untreated (DMSO control) or treated with 0.5 μM MG132, 20 ng/ml leptomycin B (LMB) or MG132 plus LMB. 24 h post-treatment cells were subjected for either (D) western blot analysis or (E) immunostaining with the indicated antibodies. (C, F-G) Nuclei and lysosomes were counterstained with Hoechst 33342 and LysoTracker Red DND-99, respectively. Nuclei in (D) were counterstained by DAPI before mounting the cells. Each panel of confocal images in (C, E-G) is representative pictures of two independent experiments. Scale bars, 5 μm. (H-I) HEK293 cells transiently transfected with plasmids expressing flag-tagged EBNA3C N-terminal (residues 1–365) and C-terminal (residues 621–992) domains in a similar experimental set up as described in (B) were subjected to subcellular fractionation as per Manufacturer’s instruction, followed by western blot analyses with the indicated antibodies. (B and H-I) GAPDH was used as reference protein for cytoplasmic fraction, while lamin A/C (B) and histone (H-I) blots were performed as nuclear reference proteins. Protein bands in (B, D, H-I) were quantified by Odyssey imager software and indicated as bar diagrams at the bottom of corresponding lanes.

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