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. 2016 Nov 28;45(6):3503–3518. doi: 10.1093/nar/gkw1144

Figure 4.

Figure 4.

JMJD6 and U2AF65 co-bind with the pre-mRNAs they regulate. (A) HEK293T cells were subjected to RIP using control IgG, anti-JMJD6 or U2AF65 antibodies, and the resultant RNAs were examined by RT-qPCR with primer sets specifically targeting to the upstream and downstream intron region relative to the alternatively spliced exon to determine JMJD6 and U2AF65 binding with pre-mRNA. The position of the alternatively spliced exon in each gene was described in Figure 2. RT: with reverse transcriptase; no RT: without adding reverse transcriptase. RIP signals were presented as fold change over IgG (RT) (± S.E.M., **P < 0.01, ***P < 0.001). (B) HEK293T cells stably expressing a control vector (CTL) or Flag-tagged JMJD6 (F-JMJD6) were subjected to RIP using anti-Flag antibody. Flag-tagged JMJD6 binding with pre-mRNAs of representative genes was shown. RIP signals were presented as fold change over control sample (RT) (± S.E.M., **P < 0.01, ***P < 0.001). (C) HEK293T cells were treated with or without UV (ultraviolet) before RIP followed by RT-qPCR as described in (A) to determine JMJD6 and U2AF65 binding with pre-mRNA. RIP signals were presented as fold change over IgG (UV) (± S.E.M., *P < 0.05, **P < 0.01, ***P < 0.001).