Figure S5.
Effect of Rags in different nucleotide loading states. (A) IF labeling of SPG15-GFP (amplified with anti-GFP), mTOR, and HA-GST–tagged RagDGDP (detected using either anti-GST or anti-HA; all Rag constructs are tandem tagged). The RagDGDP did not have any appreciable effect on SPG15, but it caused an increase (compared with nonexpressing cells) in membrane-associated mTOR in starved cells. Asterisks denote RagDGDP-expressing cells, and minus signs denote RagDGDP-negative cells. Scale bar: 20 µm. (B) Western blots of whole-cell lysates from SPG15-GFP cells transiently transfected with HA-GST–tagged RagA or RagC constructs in their GDP and GTP locked forms and labeled with anti-GST to compare relative expression levels. IF showed similar transfection efficiencies. Image is representative of two independent experiments. (C) Quantification of the fold change in SPG15-GFP spot count per object in cells expressing either RagCGDP or RagDGDP. Unlike RagCGDP, RagDGDP did not affect SPG15-GFP recruitment. The experiment was performed in biological triplicate (mean indicated; n = 3). The dotted line indicates no change. Error bars represent SEM. (D) Quantification of the fold change in mTOR spot count per object in cells expressing either RagCGDP or RagDGDP, either fed or starved conditions (1 h). All data are normalized to nontransfected fed cells. RagDGDP caused an increase in membrane-associated mTOR in both fed and starved cells. The experiment was performed in biological triplicate (mean indicated; n = 3). The dotted line indicates no change. Error bars represent SEM. (E) IF labeling of SPG15-GFP (amplified with anti-GFP) in cells that had been transiently transfected with HA-GST-RagB in either GDP- or GTP-locked forms or with HA-GST-RagA in GDP-locked form. Where indicated, cells were incubated under fed or starved conditions (1 h). RagBGTP prevents the membrane association of SPG15. The asterisks indicate cells expressing the Rag construct, and the minus signs indicate cells not expressing the Rag construct. Scale bar: 20 µm. (F) IF double labeling of mTOR in cells that had been transiently transfected with HA-GST–tagged RagAGDP. Where indicated, cells were incubated under fed or starved conditions (1 h). Note that the overexpression of RagAGDP blocks recruitment of mTOR. The asterisks indicate cells expressing the Rag construct, and the minus signs indicate cells not expressing the Rag construct. Scale bar: 20 µm.