EBNA3C modulates RASSF1A expression in a ubiquitin-proteasome-dependent manner.

A-B) EBNA3C decreased RASSF1A stability. (A) 10 million Saos-2 cells were transfected with Myc-tagged RASSF1A and Flag-tagged vector or Flag-tagged EBNA3C. 24 h post-transfection, cells were treated with 40 μg/ml Cycloheximide. (B) 10 million BJAB and BJAB10 cells were treated with 40 μg/ml Cycloheximide. At the indicated time points, the cells were harvested and lysed for western blot. The relative density (RD) of RASSF1A was quantitated to measure RASSF1A rate of degradation. (C-D) EBNA3C decreased RASSF1A stability. C) 10 million Saos-2 cells were transfected with Myc-tagged RASSF1A alone or together with Flag-tagged vector or Flag-tagged EBNA3C. 24 h post-transfection, cells were incubated with 20 μM MG132 for another 16 hours. D) 10 million BJAB and BJAB10 cells were incubated with 20 μM MG132 for 16 hours. Then cells were harvested and RASSF1A expression was detected by western blot. (E) EBNA3C enhanced RASSF1A poly-ubiquitination. 10 million Saos-2 cells were transfected with the indicated constructs. 24 h post-transfection, cells were incubated with 20 μM MG132 for another 16 hours. Then the cells were harvested and subjected to immunoprecipitation using an antibody against Myc. The inputs and immunoprecipitated samples were detected by western blot. (F) Skp2 protein was recruited by EBNA3C to mediate RASSF1A degradation. GFP-tagged RASSF1A was co-transfected with plasmids encoding EBNA3C and/or Skp2 or Skp2 truncated mutant. 24 h post-transfection, cells were harvested and lysed for western blot. The expression of RASSF1A, EBNA3C, and Skp2 was detected with specific antibodies. (G) The effect of sh-Skp2 in reducing Skp2 expression was detected in Saos-2 cells. Myc-tagged Skp2 and sh-Ctrl, or sh-Skp2 were transfected into Saos-2 cells. 48 hours post-transfection, the cells were harvested and the expression of Skp2 was detected by western blot. (H-J) Silencing Skp2 rescued RASSF1A protein expression. Skp2 knocked down BJAB H), BJAB10 I), and LCL1 cells J) were constructed by lentiviruses and selected by puromycin. 3 days later, the cells were harvested and lysed for western blot analyses. The expression levels of RASSF1A, EBNA3C, Skp2, and GAPDH were detected. The relative density (RD) of RASSF1A was quantitated and shown.