U2AF65 regulates JMJD6 binding with pre-mRNAs they co-regulate. (A, B) HEK293T cells were transfected with control siRNA (siCTL) or two independent siRNAs specifically targeting JMJD6 (siJMJD6(1), siJMJD6(2)) or U2AF65 (siU2AF65(1), siU2AF65(2)) followed by RIP with control IgG or antibody specifically against JMJD6 or U2AF65. JMJD6 (A) or U2AF65 (B) binding with pre-mRNAs of representative genes was shown as indicated. RIP signals were first normalized to input and then presented as fold change over control (siCTL) (± S.E.M., *P < 0.05, **P < 0.01, ***P < 0.001). (C) HEK293T cells were transfected with vectors expressing GFP-tagged wild-type U2AF65 (wt) or U2AF65 mutants with lysine 15, 38 or 276 substituted with arginine (K15R, K38R or K276R), followed by RIP with anti-GFP antibody. U2AF65 binding with pre-mRNAs of representative genes was shown as indicated. RIP signals were first normalized to input and then presented as fold change over control (wt) (± S.E.M., *P < 0.05, **P < 0.01, ***P < 0.001).