Figure 4.

Impaired interactions of HDAC3 variants with NCoR1/2 and KDM1A

(A) Silver-stained SDS-PAGE analysis demonstrating the protein complexes co-immunoprecipitated (IP) with FLAG-tagged HDAC3 from HEK293T cells. Tested conditions include an empty vector (EV), wild type (WT), and HDAC3 variants (p.Ala110Thr, p.Gly267Ser, p.Leu266Ser, and p.Arg359Cys). The band intensities corresponding to the NCoR complex (indicated by red arrow) and KDM1A (indicated by blue arrow) are reduced in the variant forms. Specifically, the p.Ala110Thr shows a remarkable decrease in NCoR complex band intensity (red rectangle), and all variants exhibit diminished KDM1A bands (blue rectangle).

(B) Western blot analyses confirm the differential co-immunoprecipitation of NCoR1, NCoR2, and KDM1A with HDAC3 variants, using an anti-FLAG antibody for immunoprecipitation. FLAG-tagged HDAC3 and GAPDH serve as a reference for protein levels and loading control, respectively.

(C) Quantification of co-immunoprecipitated NCoR1, NCoR2, and KDM1A, normalized to the WT HDAC3 levels, based on the Western blot data in (B). This panel quantitatively depicts the significant interaction deficits in the p.Ala110Thr and p.Arg359Cys variants with NCoR1, NCoR2, and KDM1A, despite these variants retaining deacetylase activity comparable to WT. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗∗p < 0.0001. Data are plotted as mean ± SD (n = 3/data point).