FIGURE 4.

Depletion of p115 results in complete vesiculation of the interphase Golgi, which is rescued by a construct containing both the N and C termini. A, Western blot of p115 protein levels in HeLa and RPE cells, following 4 days of either non-silencing or p115 siRNA treatment. Comparable knockdown levels and phenotypes were obtained with siRNA1 (17) and siRNA2 (36) in HeLa and RPE cells. Loading controls: actin and clathrin heavy chain (CHC). B, efficient p115 depletion results in a fragmented Golgi phenotype. C, HeLa cells were treated with the p115 siRNA for 3 days after which they were transfected for 24 h with the indicated GFP-tagged, siRNA-resistant bovine p115 constructs. Cells were fixed and processed for indirect immunofluorescence with the Golgi marker GRASP65. The rescue efficiency was quantified by scoring the Golgi morphology in 100 transfected cells. Asterisks denote fragmented Golgi in p115-depleted, untransfected/unrescued cells. Images are projected confocal Z-series. Blue, Hoechst 33342. Scale bars, 10 μm.