FIG 3.

Overexpression of IBV E alters Golgi pH. (A) The trans-Golgi pH in HeLa cells transiently expressing pHluorin-TGN38 was assessed by determining the ratio of the pH-sensitive dual-emission spectrum by flow cytometry. The cells were in buffers of known pH and contained ionophores to equilibrate the extracellular and luminal pH of the Golgi membrane. Data from a representative flow cytometry experiment are graphed. (B) Calibration curves were generated from data like those illustrated in panel A, in order to calculate the pH of cells expressing pHluorin-TGN38 alone or in combination with IBV E or E mutants. The calibration curve pictured was derived from data from 7 independent experiments (∼5,000 cells each). Error bars indicate SEM. (C) Cell emission ratios for HeLa cells expressing pHluorin-TGN38 alone and with IBV E or IBV M (control) from a representative experiment. (D) Average calculated pH values from 3 independent experiments (∼5,000 cells each). Unpaired t tests were performed in Prism at 99% confidence, with an assumption of equal variance. *, P < 0.05; ***, P < 0.001. Error bars indicate SEM.