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. 2015 Dec 2;35(48):15875–15893. doi: 10.1523/JNEUROSCI.0882-15.2015

Figure 6.

Figure 6.

Olig1 is acetylated at lysine 150 in vivo. A, B, Olig1 is acetylated at lysine 150 by CBP in HEK293T cells. Olig1-myc or Olig1-K150A-myc was cotransfected with HA-CBP plus/minus CBP shRNA A, or with p300-myc or p300DY-myc (acetylase-deficient p300; B) into HEK293T cells. Note increased acetylation in the presence of HA-CBP, which is reduced for Olig1-K150A-myc (A) or by the presence of shCBP. However, increased acetylation by P300 is comparable for WT Olig1 and Olig1 K150A-myc (B). C, The acetyl-Olig1-K150 antibody specifically recognized recombinant human Olig1 protein acetylated on lysine 150. One microgram of recombinant WT Olig1 or acetylation-deficient Olig1 mutant protein (Olig1-K150R) was incubated in vitro with purified CBP, P300, or GCN5 and their acetylation was detected by immunoblotting with acetyl-Olig1-K150 antibody, acetylated lysine antibody, or GST antibody. The activity of the different HATs was indicated by auto-acetylation of the GST-acetyltransferase detected by acetylated lysine antibody (top rows). The GST-Olig1 proteins were detected by GST immunostaining (bottom rows) and quantification of the ratio of acetylated Olig1 to total Olig1 is presented in red, normalized to the WT control. D, Olig1 was acetylated at lysine 150 by CBP in HEK293T cells, as detected by acetyl-Olig1-K150 antibody. Olig1-myc, Olig1-K150R-myc, or Olig1-K150Q-myc was cotransfected with HA-CBP into HEK293T cells. Twenty-four hours later, cells were treated with 500 nm TSA for 10 h to inhibit deacetylation before the cell lysate was immunoprecipitated with Olig1 antibody and detected with acetyl-Olig1-K150 antibody. E, Olig1 was acetylated at lysine 150 in developing mouse brain. Subcortical white matter from P3, P10, or adult samples was fractionated and endogenous Olig1 was immunoprecipitated from whole lysates or cytoplasmic or nuclear fractions and immunoblotted with acetyl-Olig1-K150 antibody. Arrowheads (red), Olig1; asterisks (black), rabbit IgG. All data represent the results from three independent experiments. Quantification of the ratio of acetylated Olig1 to total Olig1 is indicated below in red.