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. Author manuscript; available in PMC: 2014 May 9.
Published in final edited form as: Mol Cell. 2013 Apr 11;50(3):444–456. doi: 10.1016/j.molcel.2013.03.005

Figure 4.

Figure 4

3xMBT detects a novel methylation event on the lysine deacetylase SIRT1 and on candidate G9a substrates. (A) Coomassie stain of full-length recombinant GST-tagged SIRT1 and SIRT1K622R purified from Sf9 cells. (B) G9a methylates SIRT1 as lysine 622. Autoradiograph of in vitro methylation assays on SIRT1 and SIRT1K622R using recombinant G9a SET domain. (C) 3xMBT, but not 3xMBTD355N, precipitates methylated SIRT1 from cells. GST-3xMBT or GST-3xMBTD355N pull-down assays as in (Figure 2E) of NE from 293T cells expressing 3xFlag-SIRT1 or 3xFlag-SIRT1K622R and G9a or control vector. Pull-downs were probed with anti-Flag antibody to detect SIRT1. (D) 3xMBT detects G9a-methylated substrates on protein microarrays. Representative blocks of human Invitrogen ProtoArrays® methylated in vitro using GST alone (left) or GST-tagged recombinant G9a SET domain (right). Methylation was detected by probing the arrays with 3xFlag-3xMBT, followed by α-Flag antibody and species-matched fluorescent secondary antibody. Magnified regions show examples of G9a-methylated proteins detected by Flag-3xMBT. A list of all candidate G9a-methylated proteins is shown in Table S1. (E) Scatter plot comparing GST array signal-to-noise ratio (SNR) and G9a SNR for ranked ProtoArray® hits shown in Table S1.