FIGURE 2:

Lysosomal redistribution of GPP130-FM. (A) Cells were cotransfected with HA-tagged GPP130-FM and GFP-tagged Rab7 and then treated with a 1-h AP washout. Images compare GFP fluorescence from the transfected proteins and anti-HA staining, with the indicated regions expanded and presented as insets. At the 1-h time point, cells vary in their extent of residual Golgi-localized GPP130-FM. To better illustrate our analysis, the cell shown here has mostly punctae-localized GPP130-FM but equivalent results were obtained in all cells. Arrowheads mark examples of costained punctae. Bars, 10 μm. (B) The percentage of total GPP130-FM punctae counted that were positive for Rab7 is quantified for the indicated time points (mean ± SEM, n = 3, >10 cells/experiment, p < 0.05 for 0 h vs 1 or 5 h). (C, D) Blot and quantification showing level of GPP130-FM in cell extracts prepared at the indicated times after AP washout with tubulin as a loading control (mean ± SEM, n = 3). (E, F) Blot and quantification showing level of GPP130-FM in cell extracts prepared after either 0 or 5 h of AP washout or 24 h of pretreatment with pepstatin and leupeptin (+inh) followed by 5 h of AP washout (mean ± SEM, n = 3).