Supplementary Fig 2.

Detect endogenous changes of all four PTMs for Ras. (A). Serum-restricted A431 cells were stimulated with EGF for the given time period. WCL was analyzed for Ras levels. Tubulin was used as a loading control. Unstimulated and EGF treated A431 lysates were incubated with (B) APY03-beads to immunoprecipitate tyrosine-phosphorylated proteins and analyzed for tyrosine-phosphorylated Ras, (C) ASM24-beads to immunoprecipitate SUMOylated 2/3 proteins and analyzed for SUMOylated 2/3 Ras, (D) UBA01-beads to capture ubiquitinated proteins and analyzed for ubiquitinated Ras, (E) and acetyl lysine binding beads to immunoprecipitate acetylated proteins and analyzed for acetylated Ras; shown are representative westerns from N≧3 independent experiments.

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