Smad2 half-life is decreased in the presence of EBV. (A) Treatment of KM-H2 cells with cycloheximide reveals that the turnover of Smad2 is increased in EBV-positive cells relative to EBV-negative controls (KM-H2-neo). (B) Scatter plot generated from normalized densitometry values from Smad2 and actin immunoblots from cycloheximide-treated KM-H2 cells. The half-life of Smad2 in EBV-positive cells is decreased in comparison with EBV-negative controls (KM-H2-neo). (C) Immunoprecipitation from L428 HL cells stably expressing an EBNA1-GFP fusion protein using anti-GFP antibody (which pulls down EBNA1-GFP) followed by immunoblotting for either USP7 (top panel) or Smad2 (bottom panel). A positive band is observed for USP7, a known EBNA1 interactor, in the EBNA1-GFP-IP, but not in the IP with isotype control antibody. An interaction between USP7 and EBNA1 was also confirmed by IP with USP7 and subsequent immunoblotting for EBNA1 (data not shown). No bands are observed in the Smad2 immunoblot, suggesting that EBNA1 and Smad2 do not physically associate. Immunoblotting on whole lysates confirms that USP7 and Smad2 are expressed in these cells. The black dividing lines signify where images from different parts of the same immunoblot have been moved together to bring samples next to each other for direct comparison.