Product specificity of MLL3-SET wild-type and Y4884C at protein level. (A) Methylation of recombinant histone H3 protein by MLL3-SET wild-type and Y4884C alone and in the presence of complex members using unlabeled AdoMet as cofactor. After methylation, the proteins were separated by SDS-PAGE, blotted and the methylation was detected using an H3K4 trimethylated antibody (upper panel). The lower panel shows the Ponceau S-stained image of the blot. The bar diagram shows the average methylation signal from two independent experiments. The error bars indicate the standard error of the mean. (B) Global histone H3K4me3 methylation analysis from HEK293 cells. The cells were transfected with different MLL3 variant plasmids, histones were isolated, and H3K4me3 methylation was probed by Western blot. The upper image shows the H3K4me3 signal and a Ponceau S stain as loading control. The bar diagram shows the quantification from three experiments. The error bars display the standard error of the mean. The signal obtained from the MLL3 was set to 1, and the other signals were normalized accordingly.