Fig. 4.

siRNA knockdown of AUF1 has little effect on the basal level or the pH-responsive increase in PEPCK mRNA and protein. LLC-PK₁-F⁺-9C cells were transfected with 100 nM control siRNA or 100 nM AUF1 siRNA. The transfected cells were treated with either normal (pH 7.4) or acidic (pH 6.9) medium for 24 h and then harvested with lysis buffer or TRIzol. Western blot analysis was used to quantify the levels of PEPCK, β-tubulin, and AUF1 proteins. The levels of PEPCK protein at pH 7.4 (open bars) and at pH 6.9 (filled bars) were normalized relative to the level of β-tubulin. Real-time qRT-PCR was used quantify the levels of PEPCK and GAPDH mRNAs. The levels of PEPCK mRNA at pH 7.4 (open bars) and at pH 6.9 (filled bars) mRNAs were normalized relative to the level of GAPDH mRNA. The normalized data are plotted as means ± SE of triplicate samples.