Figure 4
From: A proteomic approach for the identification of novel lysine methyltransferase substrates
Validation of new SETD6 substrates. (A) ProtoArrays® were incubated with SETD6 (left) or glutathione S-transferase (GST) (right) in protein lysine methyltransferase (PKMT) reaction buffer + radiolabeled S-adenosyl methionine (SAM) overnight on a rocking platform. Scanned film was analyzed with Genepix 6.1 software. Representative magnified block images from a full ProtoArray® slide are shown for SETD6 and GST as in Figure 3B, C. (B) Venn diagram of the identified SETD6 substrates found only by fluorescence detection (group A), only by radioactivity detection (group B), and by both detection methods (group AB). The significance (P-value) of overlap for group AB was calculated using the hypergeometric distribution with a population size of 9,480 (the number of non-control proteins printed on the array). (C) Magnified ProtoArray® images of the six SETD6 candidate substrates chosen for the validation experiments. F, fluorescent-labeled SAM, R, radioactive-labeled SAM. Brackets represent the different groups defined in (B). (D) Autoradiograph of the indicated GST-tagged purified proteins that were used in the in vitro methylation assay with recombinant SETD6 followed by SDS-PAGE. The location of each protein is indicated by an asterisk. Molecular size (kDa) is shown. (E) Western blot analysis of Flag immunoprecipitations or whole-cell extracts (WCE; 2% of total) from 293T cells transfected with the indicated plasmids. f, Flag. Molecular size (kDa) is shown.