Autoregulation of Fox protein expression to produce dominant negative splicing factors

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FIGURE 6.
FIGURE 6.

Fox-1ΔE11 and Fox-3ΔE8 inhibit Fox-dependent activation of splicing. (A) The minigene DUP-E33CACNA1Cwt, FLAG-Fox-1, and increasing amounts of FLAG Fox-1ΔE11 were cotransfected in HEK293 cells. (B) A similar experiment was carried out with Fox-3 titered with Fox-3ΔE8. (Lanes −) Control transfections with the minigene only were performed at the same time. Lanes −/− were prepared similarly to lanes −, except that reverse transcriptase was omitted. (Top) The splicing of the minigene was analyzed by RT-PCR. The percent of exon inclusion is given below. (Middle) The amounts of Fox and FoxΔRRM were measured by anti-FLAG western. Anti-U1 70K Western served as a loading control. The relative FLAG-Fox levels, shown below each lane are normalized to the U1-70K content. The molar ratios of Fox to Fox-2ΔRRM are also indicated. (Bottom) The effect on splicing of exon 6 of the endogenous of Fox-2 was tested by RT-PCR. The fraction of the Fox-2 mRNA skipping exon 6 is shown below each lane.

This Article

  1. Published in Advance December 30, 2009, doi: 10.1261/rna.1838210 RNA 16: 405-416

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