Figure 4

Effect of calicivirus homologues of NV p22 on cellular protein secretion. 293T (A) or CRFK (B) cells were transfected with the vector pCMV-UTR-SEAP expressing the indicated proteins with N terminal GFP tags. Cells were washed and fresh medium was added at 22 hours-post transfection (hpt). At 24 hpt the media (containing extracellular SEAP) was collected, cells were washed 2X in cold 0.01M PBS and lysed in media containing 0.1% Triton X-100 (yielding the intracellular SEAP fraction). SEAP secretion was calculated by assaying both fractions for enzymatic SEAP activity, which were then used in the equation: SEAP secretion (%) = (SEAP_{extracellular}/[SEAP_{extracellular} + SEAP_{intracellular}]) x 100. Data are representative of at least two experiments; n = 3 for each sample; errors bars indicate standard deviations. One hundred percent of the cells expressing p22 homologue-EGFP and SEAP were viable as assessed by trypan blue exclusion (0.2% final concentration for 5 min) at 24 hpt.