FIG. 3.

Loss of LSD1 results in decreased CoREST protein levels and an increase in histone acetylation. (A) Specific antisera to the indicated proteins were used to immunoprecipitate LSD1, HDAC1, and CoREST from Lsd1^Lox/Δ3 and Lsd1^Δ3/Δ3 ES cells. Normal rabbit IgG was used as a control. Coimmunoprecipitated proteins were assessed by immunoblotting for the proteins indicated. (B) Western blotting using the indicated amounts of nuclear extract from Lsd1^Lox/Δ3 and Lsd1^Δ3/Δ3 ES cells reveals a reduction in CoREST protein in the absence of LSD1. Sin3A was used as a loading control. (C) Quantitative RT-PCR reveals that CoREST is not regulated by LSD1 through transcriptional control. CoREST mRNA levels, normalized to GAPDH, are similar in Lsd1^Lox/Δ3 and Lsd1^Δ3/Δ3 ES cells. (D) The amount of deacetylase activity associated with each immunoprecipitation was measured using a commercially available kit. Mean values are plotted (n = 3) ± standard error of the mean (SEM). (E) The methylation and acetylation status of histone H3 was detected using quantitative Western blotting. Histones were acid extracted from three different Lsd1^Lox/Δ3 and Lsd1^Δ3/Δ3 clones. The signal of specific modification was normalized to the total amount of H3 and quantitated using an Odyssey scanner (**, P < 0.01, and *, P < 0.05; Students t test).