Figure S4.

Effects of different treatments on the AP-5/SPG11/SPG15 complex versus mTORC1. (A) IF labeling of SPG15-GFP (amplified with anti-GFP) in cells that had been treated with siRNA to knock down RagA, the RagC/D GAP FLCN, or the RagA/B GATOR1 GAP subunit NPRL2. To better visualize the labeling and any effects of siRNA depletion, all cells were starved (1 h). These knockdowns have a slight effect on SPG15-GFP recruitment, but they are not as dramatic as a RagC or Ragulator knockdown. Scale bar: 20 µm. (B) Western blots of whole-cell lysates from SPG15-GFP cells treated with siRNA as indicated, with mTOR as a loading control. The blot shows mTOR labeling in a control versus FLCN knockdown, but it is representative of the loading for both knockdowns. The blots are representative of at least two independent experiments. (C) IF labeling of SPG15-GFP (amplified with anti-GFP) and mTOR in cells treated in various ways, showing an inverse relationship between the two. Starvation (1 h) increases SPG15 recruitment while preventing mTOR recruitment, while the drugs rapamycin and Torin1 lock mTOR on the membrane and prevent recruitment of SPG15. Scale bar: 20 µm. (D) IF labeling of SPG11-GFP (amplified with anti-GFP), showing that, like SPG15-GFP, its membrane association is inhibited by rapamycin and Torin1. Scale bar: 20 µm. kd, knockdown.