Fig. 3.

Characterization of LLC-ssYFP and LLC-AQP2-ssYFP cell lines. A: representative Western blot double stained for ssYFP and actin (as a loading control). Note similar expression levels of ssYFP in LLC-ssYFP and LLC-AQP2-ssYFP cell lines (lanes 3 and 4, respectively). Lanes 1 and 2 represent parental, untransfected LLC-PK₁ and LLC-AQP2 cell lines, respectively. B: Western blot double stained for AQP2, stripped and restained for actin (as a loading control). Similar AQP2 expression between untransfected parental and ssYFP-transfected cell lines indicates that YFP expression did not affect AQP2 expression (cf. lanes 3 and 4). Lanes 1 and 2 represent LLC-PK₁ untransfected parental cell lines and LLC-ssYFP, respectively. All Western blots were quantified by densitometry and are expressed as average of ssYFP- or AQP2-to-actin ratio in 3 independent experiments normalized to expression in the AQP2-ssYFP cell line. C: real-time PCR quantification of mRNA. Note similar AQP2 expression in ssYFP-expressing cells and parental LLC-AQP2 controls. LLC-ssYFP cells express ∼2-fold more ssYFP than LLC-AQP2-ssYFP cells. P0 served as an internal control, and all values were normalized to corresponding LLC-AQP2-ssYFP level. D: ssYFP fluorescence in extracellular medium of AQP2-ssYFP, as well as LLC-ssYFP, cultures grown in phenol red-free DMEM. Note steady increase to ∼4-fold over a 24-h period (normalized to 0 h), indicating constitutive ssYFP secretion within both cell lines. RFU, relative fluorescence units. **P < 0.01. ***P < 0.001.