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Figure S5. Rheb is retained in the membrane fraction in the presence or absence of growth factor treatment. (A) Serum-starved (SV) MCF7 cells were treated with 100 ng/ml EGF for 1 h in the presence or absence of 200 nM wortmannin followed by fractionation and immunoblotting with an anti-Rheb antibody. β1-integrin and LDH proteins were used as fractionation controls. (B) Rheb protein, recognized by the antibody (Cell Signaling, Inc.) can be knocked down by RNAi. HEK293 cells were transfected with Rheb siRNA (Ambion) by siPORT Amine (Ambion). Cells were harvested by RIPA buffer after 48 h of siRNA transfection followed by Rheb immunoblotting. The same blot was then reprobed with LDH and γ-tubulin antibodies, respectively, for equal loading controls.