Figure 4

HCMV sends B7-H6 to degradation. (A) Quantitative RT-PCR for B7-H6 expression in ARPE19 cells infected with HCMV US18stop, HCMV US19stop or left uninfected. Bar identities are as depicted in the figure. Relative B7-H6 mRNA quantity was normalized to the uninfected cells samples. The presented results are averages of 3 biological repeats. Error bars represent standard error of the mean. (B) Quantitative real-time PCR for US9 expression in ARPE19 cells infected with HCMV US18stop, HCMV US19stop or left uninfected. Bar identities are as depicted in the figure. Relative US9 mRNA quantity was normalized to the samples of cells infected with HCMV US19Stop. The presented results are averages of 3 biological repeats. Error bars represent standard error of the mean. (C) Western blot of HCMV infected ARPE19 cells overexpressing FLAG-tagged B7-H6. The upper row presents a blot with an anti-FLAG antibody. The lower row presents a blot with anti-human Vinculin, used as a loading control. All samples derived from the same experiment were processed in parallel. One representative experiment out of three is presented. (D) Quantification of the results presented in C. Averages of three independent experiments are presented. Error bars represent standard deviation. (E) Western blot of HCMV infected ARPE19 cells overexpressing FLAG-tagged B7-H6 and treated with DMSO (10 μl/ml), Epoxomycin (Epox, 10 μM), Bortezomib (BTZ, 500 μM), Concanamycin A (CCMA, 50 nM), Leupeptin (Leu, 500 μM) or left untreated (PBS). The upper row presents a blot with an anti-FLAG antibody. The lower row presents a blot with anti-human Vinculin, used as a loading control. The presented blots were cropped and the background was adjusted for clarity. A student’s t test was performed in order to evaluate significance. *p-value < 0.05. **p-value < 0.005. ***p-value < 0.0005. N.S. = non-significant differences.