Fig. 6

CHD-3 restricts the LET-418-dependent MES-2/Polycomb recruitment to chromatin. a Global levels of LET-418 and H3K27me3 proteins in whole embryonic extracts of wild-type or chd-3(eh4) null worms treated with control- or let-418(RNAi). Upper panel: western blots; lower panel, quantification of the western blot, represented as relative intensity normalized to actin levels. b Subcellular fractionation experiment of soluble/cytosolic, nucleoplasmic, and chromatin fractions in embryos of the same genotypes as (a). LET-418, MES-2::EGFP::3xFLAG levels were assessed by western blotting, and fractionation was verified with proper cytosolic GAPDH versus nuclear total H3 separation (upper panels). Lower panels western blot quantification, normalized to GAPDH (soluble fraction) or to H3 levels (nucleoplasm and chromatin fractions). All fractions were loaded with equal protein amounts, and the selected western blots were exposed for the same duration. c Anti-LET-418 immunoprecipitation identifies MES-2::EGFP::3xFLAG as a LET-418 interactor in embryonic extracts. Western blots of LET-418 and MES-2::EGFP::3xFLAG in embryos expressing one or the two constructs (±) and immunoprecipitated for Mock (no antibody) or LET-418-binding proteins. d chd-3 depletion does not disrupt the LET-418/MES-2 interaction. Anti-LET-418 immunoprecipitations performed in wild-type (+) or chd-3(eh4) (Δ) embryos and western-blotted for the indicated proteins. Mock and LET-418 IP western blots were selected at an identical exposure time. e Tagged CHD-3::StrepIII::HA interacts with MES-2::EGFP::3xFLAG in embryonic extracts. Strep-tactin pull-downs were performed in embryos expressing or not the CHD-3::strepIII::HA construct (±), and the presence of both tagged protein was detected by western blotting analysis. Mock IP: agarose resin only