Figure 2.
Effects of ORFV-infection on expression and intracellular transport of MHC I . (A) MHC I- and GAPDH-specific RT-PCR was performed as described in Methods. After gel electrophoresis the amplicon band intensities were quantified by densitometry and their calculated ratios are indicated below each gel lane. The transcription rate of MHC I was not affected significantly by ORFV infection. (B, upper panel) ORFV infection affects carbohydrate trimming of MHC I. Infected (+; m.o.i. 2.0) or not infected (−) cells were labelled with Trans-35 S-Label, lysed at 12, 24 and 36 hpi, and MHC I was immunoprecipitated with W6/32 antibody. The immunoprecipitates were digested with Endo H before separation by SDS-PAGE. Fluorographs were analyzed using GelEval 1.32 software (FrogDance Software). Endo H-resistant, -sensitive and partially Endo H-resistant MHC I forms are indicated. (B, lower panel) Infected (+; m.o.i. 1.0) or not infected (−) cells were lysed at 12 hpi, digested with Endo H and analyzed by Western blots probed with anti-MHC I mAb LY5.1.