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Figure S2. Depletion of Gαi3 impairs EGF-stimulated Akt activation. HeLa cells were treated with scr or Gαi3 siRNA, serum starved for 6 h, and stimulated with 100 nM EGF for 0–30 min. Lysates were separated by SDS-PAGE and immunoblotted for total Akt (tAkt) or phospho-Akt (pAkt). The ratio of pAkt to tAkt is expressed in arbitrary units and displayed below as bar graphs. Controls (scr siRNA) had robust Akt activation at 5 min followed by rapid down-regulation within 15–30 min, whereas Gαi3-depleted cells had a blunted response with an ∼60% decrease in EGF-stimulated peak Akt activation.