Fig. 5

GST-fused AsxETSI-2 interacts with E(z) in embryo nuclear extracts. a Alignment of the amino acid sequence of wt and Asx³ mutant indicates deletion of ETSI-2 and TSI sites in the COOH terminal region, which may disturb the levels of H3K4me3 and H3K27me3. b SDS–PAGE (13%) analysis of purification of E. coli-expressed GST fusions of ETSI-2 domains. c The interaction of ETSI-2 with endogenous E(z) was assayed using extracts prepared from 6- to 18-h-old embryo nuclei. Fractionation of extracts on a Bio-Rex70 column by a step gradient from 0.1 to 0.85 M NaCl elutes E(z) at 0.1 M NaCl fraction as opposed to Trx which elutes at 0.85 M NaCl. d GST-fused ETSI-2 was mixed with the 0.1 M NaCl-containing E(z) fraction in a pull-down assay, using GST as a negative control. The pull-down products were analyzed by 7.5% SDS–PAGE and probed by anti-E(z) antibody in a western blot assay. Lanes (1) pre-marked molecular weight markers (BioRad); (2) 10% input of the pull-down fraction; (3) GST/BR70-0.1 fraction pull down; (4) GST-ETSI-2/BR70-0.1 fraction pull down. The lower band in lane 4 is likely a breakdown product of E(z)