a. Schematic of the procedures to isolate the UTX (WT or ΔcIDR) and MLL4f proteins for in vitro histone methylation (HMT) and imaging analyses. Note that all HMT assays involved endogenous MLL3/4 associated with isolated UTX. b. Immunoblotting for UTX in the UTX (WT or ΔcIDR) pulldowns, along with indicated ng of purified EGFP-UTX (1-848). This allowed an estimate of the concentration of WT-GFP or ΔcIDR-GFP to be ~40 nM in the pulldowns. c. Fluorescence images of the UTX (WT or ΔcIDR) pulldowns in the condensation buffer, with ~25 nM UTX protein and 150 mM NaCl. d. Immunoblotting for the indicated proteins in the isolated UTX (WT or ΔcIDR) complexes. e. Representative results of in vitro HMT assays using the UTX (WT or ΔcIDR)-GFP pulldown. UTX and indicated histone modifications at indicated time points of HMT assay were determined by Immunoblotting. Total Histones are shown by Ponceau S staining. Right, Relative H3K4me1 increase (from time 0) is plotted as mean from 2 independent experiments. f. Representative results of in vitro HMT assays using the UTX (WT or 555*) pulldown. Top, immunoblotting by indicated antibodies and Ponceaus S staining for total histones in HMT assay. Bottom, H3K4me1 activity was determined as relative H3K4me1 increase from preexisting methylation (in control) and plotted as mean ± SD. n = 3 independent experiments. P values by two-sided t-test. g. Immunoblotting using anti-FLAG antibody for the isolated UTX (WT or ΔcIDR) complexes, MLL4f-mCherry, or their mixture as indicated. UTX (WT or ΔcIDR) and MLL4f all had FLAG tag. h. Fluorescence microscopy images of indicated protein complex, either as individual protein (top row) or mixed as indicated (bottom two rows) in the condensation buffer. The concentration of each protein in the mixture was the same as that of the corresponding individual protein shown on top row. i. Immunostaining for Myc (for Myc-tagged UTX), H3K27me3, and DAPI in COS-7 cells transfected with indicated UTX WT or mutant construct. Cells with UTX overexpression (indicated by arrows) were scored for the signal intensity of H3K27me3 and quantified in j. j. Quantification of the cells showing Decrease (↓), no change (-), or increase (↑) of H3K27me3. Shown are the percentage of cells showing the indicated changes in one out of a total of five biological repeats. In parenthesis is the number of cells showing the changes/total number of cells scored. In each repeat, around 100 cells were scored for each sample.