Characterization of Slc12a6 conditional KO mouse lines. A, Tail-clasping test done on Slc12a6^Δ18/Δ18 and nsSlc12a6^Δ18/Δ18 KO lines at 3–4 weeks of age, compared with Slc12a6^flox/flox transgenic mice and WT littermates. Resting appearance of Slc12a6^Δ18/Δ18 and nsSlc12a6^Δ18/Δ18 mice at 12 months of age exhibiting extensive hindlimb weakness, movement disorganization, and kyphosis (curvature of the upper spine). B, A representative RT-PCR performed on brain mRNA isolated from WT and Slc12a6^Δ18/Δ18 mice using primers designed from exon 17 (forward) and exon 20 (reverse). A band of ∼616 bp is seen in the WT, whereas a smaller fragment of ∼450 bp is seen in the Slc12a6^Δ18/Δ18 mice (n = 3). C, D, A representative Western blot analysis of brain lysates isolated from Slc12a6^Δ18/Δ18 (C) and nsSlc12a6^Δ18/Δ18 (D) KO lines and their heterozygous and WT littermates (n = 3), and probed with anti-KCC3 and anti-actin antibodies. A truncated protein with a molecular weight of ∼75 kDa is weakly visible in the heterozygous and KO animals (arrowheads). E, A representative Western blot analysis of kidney lysates from nsSlc12a6^Δ18/Δ18 and WT littermate mice using the same antibodies as in C and D, showing reduced KCC3 levels, but absence of the truncated KCC3 (n = 3).