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. 2021 Jan 8;49(10):5407–5425. doi: 10.1093/nar/gkaa1262

Figure 3.

Figure 3.

The EZL1 complex interacts with the nuclear RNAi component EMA1. (A) Co-localization of the EZL1 complex and EMA1. RNF1-HA cells were stained with anti-HA (green) and anti-EMA1 (red) antibodies, and counterstained with DAPI (blue). Representative images of several conjugation stages (at 3, 6, 8, 10 and 14 h post-mixing) are shown: meiosis, zygotic divisions, MIC/MAC differentiation, early anlagen (developing MAC) formation and late anlagen development. The schematic illustrates the typical nuclear morphology. Parental MAC (PM): green arrowhead; developing MAC (AN): green arrows; MIC (Mic): white arrowheads; old MAC (OM): white arrow. (B) A schematic of crosslinking IP. Cells were crosslinked with formaldehyde, and the solubilized nuclear fraction was used as IP input. (C) Co-IP of NUD1 and EMA1 in tagged cells. WT and various tagged cells were processed for crosslinking IP with the anti-HA antibody. The anti-EMA1 (top) and anti-NUD1 antibodies (bottom) were used for immunoblotting. Note the slight up-shift in the migration of NUD1-HA compared with endogenous NUD1 (arrowhead). (D) Co-IP of NUD1 and EMA1 in WT cells. The anti-NUD1 antibody was used for crosslinking IP; the anti-EMA1 (top) and anti-NUD1 antibodies (bottom) were used for immunoblotting (signal indicated by block arrows; the background band in the NUD1 panel corresponds to the IgG heavy chain); pre-bleed IgG (IgG) as negative control. (E) Reciprocal IP of NUD1 and EMA1. WT cells were processed for crosslinking IP with the pre-bleed IgG (IgG), anti-EMA1, and anti-NUD1 antibodies, respectively; the anti-EMA1 (top) and anti-NUD1 antibodies (bottom) were used for immunoblotting. (F) Nucleic acid-independent association of NUD1 and EMA1. After crosslinking IP, the anti-HA agarose was treated with RNase A or DNase I before wash and elution; no nuclease treatment (–) as the control. The anti-EMA1 (top) and anti-NUD1 antibodies (bottom) were used for immunoblotting. (G) Association of NUD1 and EMA1 in various mutants. WT and the designated mutants were processed for crosslinking IP with the anti-NUD1 antibody. The anti-EMA1 (top) and anti-NUD1 antibodies (bottom) were used for immunoblotting.