Preserved Ncc mRNA transcripts but diminished Ncc protein expression in the kidneys of Ncc T58M knock-in mice. (A) RT-PCR analysis of Ncc mRNA using primers flanking mutation sites (exon 1) (upper panels). Quantitative gene expression of Ncc in WT, heterozygous (He), and homozygous (Ho) Ncc T58M knock-in mice (n=6/group) are presented as percentage of control (control = 100%) (lower panel). (B) An antibody recognizing the amino terminal portion (amino acids 73–88) of Ncc is applied for detecting Ncc expression (upper panel). Semiquantitative immunoblot of Ncc in crude membrane fractions from WT, He, and Ho mice (middle panels) and its semi-quantification by densitometry analysis (lower panel) (n=4/group). (C) Immunofluorescence staining for Ncc. Representative immunofluorescence micrographs of Ncc in the cortex of WT, He, and Ho mice. *P<0.05 versus WT comparison. Original magnification, ×200 and ×1000 in C.