Subcellular localization of pUL138-eGFP and the effect of protease inhibitors. (A) Subcellular localization of pUL138-eGFP with LC3 and LAMP2 in T2.B35 stable transfectants. T2.B35 cells stably expressing pUL138-eGFP fusion protein were costained with anti-GFP in AlexaFluor-488 and anti-LC3B or anti-LAMP2 in AlexaFluor-546. Cells were either untreated or pretreated with chloroquine (80μM) for 60 hours. Colocalization of pUL138-eGFP (shown in green) and LC3 or LAMP2 (shown in red) is indicated in white. Blue represents nuclei staining with 4,6-diamidino-2-phenylindole (DAPI). Scale bars represent 5 μm. Original magnification ×64 (Olympus IX70 microscope, DeltaVision microscopy system). Image deconvolution and colocalization analysis performed with softWoRx. Right panels: scatter plots and degree of colocalization of pUL138-eGFP with LC3 or LAMP2 by Pearson coefficient of correlation. Results are representative samples from 3 independent experiments. (B) Effect of protease inhibitors on pUL138 TAP-independent antigen presentation. T2.B35 cells were pretreated with protease inhibitors and infected with AdUL138. Data shown are from 3 independent experiments performed in triplicate samples (mean ± SEM). **P < .05 (2-tailed Student t test). The mean absolute percentage of T-cell response to reference cells was 11.3%.