FIGURE 7:

(A) HeLa cells expressing the ManII-GFP RUSH reporter were transfected with the indicated siRNA. After 72 h, cells were treated with biotin to release the reporter from the ER. Cells were fixed at indicated time points, and the number of punctate structures (transport complexes) was determined using ImageJ. Bottom, representative images; top, statistical evaluation of three independent experiments (normalized to time point 0 min). (B) HeLa cells were transfected with the indicated siRNA. After 48 h, cells were transfected with the VSVG-RUSH construct (encodes for GFP-tagged VSVG and streptavidin-Ii). After further 24 h, cells were either lysed directly (T0) or treated with biotin (to release VSVG from the ER), lysed after 10 (T10) and 20 min (T20), and lysed subsequently. Cell lysates were either left untreated (–) or digested with endoglycosidase H (+). R and S, positions of the endoglycosidase H–resistant and –sensitive bands, respectively. (C) Effect of PLCG1 depletion on the distribution of ERGIC-53 in HeLa cells. (D) The first two bars represent the quantification of the experiments described in C (three independent experiments ± SD). The remaining bars represent quantifications of the number of peripheral ERGIC-53 punctae from cells transfected with the indicated mutants of PLCG1 24 h before fixation and staining. Asterisks indicate statistically significant differences. Asterisks indicate statistically significant differences from control (** p < 0.01).