Figure 1

SILAC-based proteomic analysis of membrane protein changes induced by eIF6 overexpression. A) Schematic representation of SILAC-based proteomic workflow. B) 10 micrograms of protein whole cell extracts isolated from A2780 transfected either with pcDNA3.1 and pcDNA3.1-eIF6 were separated by SDS-PAGE and transferred to a PVDF membrane. Bands relative to eIF6 and tubulin (loading control) were detected with respective antibodies and analyzed by densitometry using Quality-One software (Bio-Rad laboratories, Richmond, CA). The X-axis shows the relative intensity of eIF6/tubulin; one representative experiment out of three is shown. C) Equal amounts of protein whole cell extracts isolated from control (pcDNA3.1) and eIF6-overexpressing (pcDNA3.1-eIF6) cells were mixed and subjected to native membrane purification. 10 micrograms of whole cell extract (WCE), soluble (S) and membrane (M) fractions were analyzed by western blotting. Antibodies against calnexin and GAPDH were used as markers of membrane and soluble fractions, respectively. One representative experiment out of three is shown.