Figure 5. Acinus stimulates the use of the weaker alternative 5′ splice site of endogenous RARβ in a RA-dependent manner (A) and endogenous Bcl-x in a RA-independent manner.

293A cells were transfected with V5-Acinus-L, V-5Acinus-S’ or V5 empty expression vector DNA. Twenty-four hr after transfection, cells were treated with either ethanol or 10⁻⁶ M RA. After an additional 24 hr, RNA was isolated and RT-PCR was performed using primers that detect both spliced forms as indicated by the arrows in the schematic diagram of RARβ (A) or Bcl-x (B). The products were separated by gel electrophoresis, stained with SYTO 60 and quantitated using LI-COR Odyssey Infrared Imaging System. Note that for RARβ in Panel A, visualization of the PCR products in the ethanol treated samples required 35 PCR cycles while in the RA treated samples 30 PCR cycles were sufficient. The isoform ratio of the empty vector control treated with ethanol was set to 1. Values represent mean ± SD from 3 independent experiments. V, V5 empty vector; L, V5-Acinus L; S’ V5-Acinus-S’; Eth, ethanol; RA, retinoic acid. * p < 0.05, ** p < 0.01, *** p < 0.001, unpaired t test.