Figure 6.

PLA2G6-A controls tubulation of the ERGIC under conditions where Arf1+Arf4 are not depleted. (A) HeLa cells stably expressing GFP-ERGIC-53 were incubated or not at 16°C for 3 h and rewarmed or not in the presence or absence of BEL. Cells were then stained with anti-PLA2G6-A. Arrows point to GFP-ERGIC-53 tubules. Filled arrowheads point to PLA2G6-A spots colocalizing with ERGIC clusters. Empty arrowheads indicate PLA2G6-A spots connected to ERGIC tubules. Bottom panels are magnifications of the highlighted regions. (B) Cells were cotransfected with pDsRedT1 (RFP) and either a control shRNA or a shRNA targeting PLA2G6-A. Cells were then rewarmed from 16°C as described above. PLA2G6-A knockdown cells were identified by RFP signal. The arrowhead points to ERGIC tubules. Insets are magnifications of GFP-ERGIC-53 channel in the highlighted regions. (C) PLA2G6-A knockdown cells treated as described in B were counted as described in Figure 3F. Data are represented as mean ± SD from five to six experiments. The p value was calculated using a Student's t test. (D) Cells were either mock transfected or transfected with L-iPLA₂. Arrows point to GFP-ERGIC-53 tubules. Arrowheads point to PLA2G6-A spots localizing to ERGIC clusters and connected to ERGIC tubules. Insets are magnifications of the highlighted regions. Bars, 5 μm.