Fig. 4.

Recruitment of MS2cp-p40AUF1 is also not sufficient to impart a pH-responsive stabilization of βG-PCK2-MS2 mRNA. A: LLC-PK₁-F⁺-9C cells that stably express the βG-PCK2-MS2 reporter RNA were transiently transfected with 2 μg of pFlag-MS2cp-p40AUF1 and maintained in normal (pH 7.4) medium before being harvested with lysis buffer. Western blot analysis was performed to assess the expression of the endogenous and chimeric forms of AUF1. B: half-life analysis of the βG-PCK2-MS2 reporter RNA in LLC-PK₁-F⁺-9C cells that transiently express MS2cp-p40AUF1. Cells were maintained in minimal Dox (100 ng/ml) until 70% confluent. Following transient transfection with 2 μg of expression plasmid, the cells were maintained in normal (pH 7.4) medium minus Dox for 48 h. The cells were then treated with normal (red) or acidic (green) medium for 24 h. RNA was isolated at various times after addition of 1 μg/ml Dox to arrest transcription. The relative levels of β-globin and GAPDH mRNAs were quantified by RT-qPCR. The log of normalized data was then plotted against the time after Dox addition. The reported data are means ± SE of triplicate samples.