Fig. 5

Localization of SLC12A1 in the affected fetal kidneys in Japanese Black cattle. a Total RNA was extracted from kidneys of the unaffected (blue dots) and affected (magenta dots) fetuses at approximately 160 days of gestation, and the unaffected adults for real-time quantitative PCR. Relative gene expression levels in the different samples are shown as mean quantities relative to the value observed in the adult kidney (dotted line). b, c, d, e Localization of SLC12A1 in the kidneys of unaffected (b, d) and affected (c, e) fetuses at approximately 160 days of gestation. Sections were immunostained with anti-SLC12A1 antibody (magenta) and counterstained with DAPI (blue). Images b, c and d, e show the renal cortex and medulla, respectively. b, c Distal convoluted tubule indicated by arrow. d, e Thick limbs of the loop of Henle are indicated by arrows. e Dilated thick limbs of the loop of Henle in affected fetuses are indicated by asterisks. f Line plots of fluorescence intensities from apical to basal side of distal convoluted tubule in the unaffected (blue line, b) and affected fetuses (magenta line, c). g Membrane and cytosolic fractions were prepared by centrifugation from the unaffected (lanes 1 and 2) and affected (lanes 3 and 4) fetuses at 100,000 × g and the immunoblot (IB) was performed using anti-SLC12A1 antibody. Scale bars: b, c = 20 μm; d, e = 10 μm