Supplemental data
Supplemental Figure 1. Characterization of opossum kidney cell line stably transfected with rat AQP1 (AQP1-OKC). (A) AQP1-OKC form cilia and microvilli as demonstrated by staining of microtubules (red) and actin filaments (green). Magnification: scale bar = 10 μm. (B) Western blot of AQP1-OKC (lane 1), untransfected OK cells (OKC; lane 2), and mouse kidney BBM fraction (lane 3). Typical AQP1 signal is observed in lane 1 and 3 at 28 kDa (unglycosylated AQP1) and 35-50 kDa (glycosylated AQP1).
Supplemental Figure 2. Calcein fluorescence quenching. (Left) Calcein-loaded AQP1-OKC were stimulated for 1 hour with FSS, 8-bromo-cGMP or 8-bromo-cAMP, and calcein fluorescence was monitored. Representative curves show the time-course of changes in calcein fluorescence in response to a switch in solution osmolality from 500 to 150 mOsmol. (Right) AQP1-OKC grown in flow chambers were calcein-loaded and various rates of fluid-shear stress were applied for 1h. Representative curves showing the time-course of changes in calcein fluorescence in response to a switch in solution osmolality from 500 to 150 mOsmol.
SupplementaI Table 1. AQP1 mRNA abundance. AQP1 mRNA was analyzed by real time PCR. ¨¨CT values of AQP1 and the house keeping gene, GAPDH, of Lrp2fl/fl;apoECre, Clcn5fl/y;villinCre, Clcn5+/y, and Cclcn5-/y mice are shown. Values are presented as means ± SD; n = 6. AQP1 mRNA is not different between groups.
Published in "Journal of the American Society of Nephrology doi: 10.1681/ASN.2014020148, 2014"
TubulinPhalloidin Tubulin Phalloidin AQP1 45 kDa 35 kDa 28 kDa
-AQP1-OKC OKC BBM mouse
B
A
-10 0 10 20 30 40 50 60 70 80 90 0 0.8 1.6 2.4 3.2 4 4.8 5.6 6.4 7.2 8
Supplemental Figure 2
-5 0 5 10 15 20 25 0 1.35 2.7 4.05 5.4 6.75 8.1 con FSS cGMP cAMP 0 dyn/cm2 0.5 dyn/cm2 1 dyn/cm2 2 dyn/cm2 4 dyn/cm2 Calcein fluorescence Calcein fluorescenceTime in sec Time in sec
500 150 mOsmol 500 150 mOsmol
Supplemental Table 1
Calculations of AQP1 mRNA ǻǻCT Lrp2fl/fl vs. Lrp2fl/fl;apoECre 0 ± 1.2% vs. –0.02 ± 0.8% Clcn5fl/y vs. Clcn5fl/y;villinCre 0 ± 0.3% vs. –0.04 ± 0.26%