JMJD6-mediated lysine hydroxylation of U2AF65 is involved in alternative splicing regulation. (A) HEK293T cells were transfected with control siRNA (siCTL) or two independent siRNAs specifically targeting U2AF65 (siU2AF65(1), siU2AF65(2)) in the presence or absence of a control vector or vectors expressing wild-type U2AF65 (wt) or its mutants with substitution of lysine 15, 38 or 276 to arginine (K15R, K38R or K276R), followed by immunoblotting (IB) with anti-U2AF65 (upper panel) or anti-GFP antibody (bottom panel).(B) Pie chart showing alternative splicing events regulated by U2AF65, both exon inclusion and skipping (FC≥2), examined through RASL-Seq analysis (expanded version) using RNA samples described in (A). Data shown was from siU2AF65(1). (C) Scatter plot showing the isoform ratio (short versus long, log2) of all detectable alternative splicing events in RASL-Seq (expanded version) when knocking down U2AF65. (D, E) Far left panels: Pie chart showing exon inclusion (D) or skipping (E) regulated by U2AF65, including the ones could be rescued by U2AF65 protein (referred to as exon inclusion (D) or skipping (E) truly regulated by U2AF65) and the ones could not; Right three panels: Pie chart showing exon inclusion (D) or skipping (E) truly regulated by U2AF65, including the ones were dependent on K15, K38 or K276 as indicated, and the ones were not.