Supplementary Fig 3.
Detection of all four PTMs for c-Fos. Serum-restricted A431 cells were either unstimulated or stimulated with EGF for 60 minutes prior to lysis with BlastR lysis buffer. Whole cell lysate (WCL) was analyzed for c-Fos levels (lanes 1,2). Ubiquitin control beads (CUB02) were used to immunoprecipitate non-specific binding proteins (lanes 3,4). Ubiquitin binding beads (UBA01) were used to immunoprecipitate ubiquitinated proteins (lanes 5,6). Acetyl lysine binding beads (15E12) were used to immunoprecipitate acetylated proteins (lanes 7,8). Phospho-tyrosine binding beads (APY03) were used to immunoprecipitate tyrosine-phosphorylated proteins (lanes 9,10). SUMO 2/3 binding beads (ASM24) were used to immunoprecipitate SUMOylated 2/3 proteins (lanes 11, 12). IgG binding control beads were used to immunoprecipitate non-specific binding proteins (lanes 13,14). All samples were separated by SDS-PAGE and analyzed by western immunoblotting using a c-Fos antibody to identify changes in c-Fos PTMs in response to EGF. Shown is a representative western from N≧3 independent experiments.