Figure 1: Properties of M8 motif interactome. (A) The M8-cis regulatory element is shown in bold letters, whereas the point mutations designed to disrupt DNA binding are highlighted in red. (B) Specific M8 interaction partners cannot be identified by one-dimensional SDS-PAGE and colloidal coommassie staining comparing the eluted material from the affinity columns carrying either a M8 wild-type (WT) or mutant (MT) sequence. (C) Scheme of biotinylated wild-type and mutant M8 sequences used for immobilization. The PstI restriction site (CTGCAG) is indicated (italics). (D) Immunoblot analysis of selected M8-specific interaction partners confirms results from the SILAC analysis. Equal amounts (20% of total) of input (IN) and flow-through (FT) as well as eluted (Elution) material (20% of total) from the M8 wild-type (WT) and mutant (MT) columns, respectively, were analyzed by Western-blot with antibodies directed against TFCP2 and TFCP2L1. The Poly [ADP-ribose] polymerase 1 protein (PARP1) serves as a control for equalized total protein loading amounts and unspecific DNA background binding. (E) SILAC ratio distribution of all proteins identified at least once in a forward and reverse experiment. Proteins containing at least two peptides with log2 SILAC ratios (lysine-2H4 or arginine 13C6 vs lysine-1H4 or arginine 12C6) greater than 1.0 are initially considered specific. Likewise, in reverse experiments (see text) the log2 of inverted ratios was used. Scatter plot of SILAC ratios of all proteins identified at least once in a forward and reverse experiment. The mean ratios of proteins which were found in at least one forward and one reverse experiment were plotted. Proteins enriched on the wild-type bait were initially considered as positive hits and further analyzed by statistics.