Integration of Golgi trafficking and growth factor signaling by the lipid phosphatase SAC1

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Figure S2. Growth-dependent SAC1 shuttling from the Golgi to the ER is independent of PI3 kinase and mTOR in quiescent human fibroblasts. (A) Cells were grown in the presence of low (0.3%) FCS for 48 h and then stimulated with 15% FCS in the presence of a 100 nM PI3 kinase inhibitor (wortmannin) or 5 µM mTOR inhibitor (rapamycin). Cells were fixed, permeabilized, stained with anti-SAC1 antibodies, and then analyzed by immunofluorescence microscopy. Bar, 15 µm. (B) Efficiency of PI3 kinase and mTOR inhibition was controlled by immunoblotting with antibodies against p-Akt, p-S6-kinase, or GAP-DH as indicated. (C) p38 MAPK inhibition does not cause a general block in COP-I–dependent retrograde traffic. NIH3T3 cells were electroporated with cDNAs to express GFP-CD4 chimeras containing either a functional KKLY ER retrieval motif or a mutated AALY sequence. Cells were serum starved for 24 h and then stimulated with 10% serum for 1 h in the presence or absence of 10 µM SB203580. Cells were fixed, permeabilized, and stained with polyclonal anti-SAC1 antibodies.