FIG 2.

IBV infection alters Golgi pH. (A) Vero cells stably expressing pHluorin-TGN38 were evaluated by indirect immunofluorescence microscopy. Cells were labeled with rabbit anti-golgin-160 and mouse anti-GFP, followed by Alexa 546-conjugated anti-rabbit IgG and Alexa 488-conjugated anti-mouse IgG. (B) Vero cells stably expressing pHluorin-TGN38 were used to assess the pH of the trans-Golgi network 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. (C) Calibration curves were generated from data like those illustrated in panel B, in order to calculate the pH of cells infected with IBV or uninfected cells. The calibration curve pictured was derived from data from 3 independent experiments (∼10,000 cells each). Error bars indicate standard errors of the means (SEM). (D) Cell emission ratios for Vero cells infected or mock infected with IBV and stably expressing pHluorin-TGN38 from a representative experiment. (E) Average calculated pH values from 3 independent experiments (∼10,000 cells each). Unpaired t tests were performed in Prism at 99% confidence, with an assumption of equal variance. ***, P < 0.001. Error bars indicate SEM.