Fig. 1. Interactome analysis of Rpd3 in MB neurons.

a Thermogenetic activation of MB neurons. GFP fused to the nuclear localization signal (nlsGFP) and dTRPA1 was induced in MB neurons using MBsw. The brains were immunostained with anti-GFP (green) and anti-pERK (magenta) antibodies, and DAPI (blue). The images are representative of experimental replicates (n = 3, 4, 4, and 4). Scale bar: 10 μm. b Purified Rpd3 proteins from MB neurons. Rpd3::FLAG-HA was expressed by MBsw, together with dTRPA1. The flies were heat-shocked at 35 °C for 1 h, and the heads were used for tandem-tag affinity purification. The proteins were visualized by silver staining. Asterisks indicate the IgG heavy and light chains. c The amount of the representative CoRest peptides identified in the LC-MS/MS analysis. See more details in Supplementary Table 2. (Top) The vertical gray bars indicate the exons, and the horizontal lines indicate the region of the introns. The regions targeted by the anti-CoRest antibody and RNAi were indicated as black bars. d Expression of CoRest-F and CoRest-C. (Left) The flies expressing nlsGFP by MBsw were used to immunostain CoRest-F with the anti-CoRest antibody which detected the N-terminal domain of CoRest (magenta). (Right) The genomic region of CoRest-C was cloned, tagged with myc, and inserted into a different region of the genome. Flies also expressing nlsGFP by MBsw were immunostained with the anti-myc antibody (magenta). The images are representative of three experimental replicates. (Upper two panels) Scale bar: 10 μm. (Lower three panels) Magnified view depicted as white squares. Scale bar: 2 μm. e Working hypothesis of the complex compositional change in Rpd3/CoRest. Source data are provided as a Source Data file.