Figure S3.

Knockdown of small GTPases or their modifying enzymes. (A) IF labeling of SPG15-GFP (amplified with anti-GFP) in cells that had been treated with siRNA to knock down Rab7a, Rab9a, or both together; Arl8a/Arl8b; and CCZ1. This was compared with control cells transfected with a nontargeting siRNA under fed conditions. Fig. 3 shows the same treatments in starved cells. Scale bar: 20 µm. (B) IF labeling of SPG15-GFP (amplified with anti-GFP) in cells that had been transfected with TBC1D5, a GAP for Rab7, tagged with mCherry. Note that the overexpression of mcherry-TBC1D5 causes the loss of Rab7 from membranes but has no apparent effect on the localization of SPG15-GFP. Scale bar: 20 µm. (C) Localization of SPG15-GFP (amplified with anti-GFP) and Rab7 under control conditions or after treatment with either a PI3K inhibitor (100 nM wortmannin; 1 h) or a PI5K inhibitor (1 µM YM201636; 1 h). Scale bar: 20 µm. kd, knockdown.