Figure 3
From: A proteomic approach for the identification of novel lysine methyltransferase substrates
Identification of new protein lysine methyltransferase (PKMT) substrates using the fluorescent detection approach: (A) A diagram of the procedure used to define a positive hit. See text for detailed description. (B, C) ProtoArrays® were incubated with (B) SET domain-containing SETD7 and (C) SETD6 in PKMT reaction buffer overnight at 30°C, probed with a pan-methyl antibody, then followed by washes and incubation with an Alexa Fluor 647 secondary antibody. Arrays were then scanned (Axon Genepix 4000B; Molecular Devices) and analyzed using Genepix 6.1 software. Representative magnified block images from a full ProtoArray® slide are shown for SETD6, SETD7, and glutathione S-transferase (GST; negative control). The enlarged region shows specific examples of substrate methylation by the different enzymes, and their relative location on the arrays. Graphs below images show the calculated signal-to-noise ratio (SNR) for individual substrates of each enzyme (red; 321 substrates for SETD7 and 118 for SETD6), compared with GST (blue). SNR was calculated based on three independent experiments for GST and two independent experiments for both SETD7 and SETD6. SD, standard deviation.