Construct design and validation of the Knock out mice.

A. Knock out DNA constructs design. The 138 bp starting with the first ATG of the coding sequence of the gene up to the end of the deletion in patients, 300 bp into the adjacent 1^st intron were replaced by a β-gal- Neo cassette of 5600 bp. B. PCR genotyping of the mice. The primers amplifying each of the DNAs are exclusive for that DNA. CaM KMT^-/-: KO, CaM KMT^+/+: WT, CaM KMT^+/-: HET, 1Kb plus DNA ladder (Thermo Scientific): Ma. C. Validation of transcription. RT-PCR on kidney RNA using primers in the 1^st and 2^nd exons of CaM KMT. CaM KMT^-/-: KO, CaM KMT^+/+: WT CaM KMT^+/-: HET, negative control no DNA: n.c, 1kb plus DNA ladder (ThermoSCIENTIFIC):Ma. D. Validation of translation. Liver and kidney homogenates of CaM KMT^-/-:-/-, CaM KMT^+/+: +/+ were analyzed by Western blotting with purified CaM KMT polyclonal antibody [11]. Adjacent lanes on the same gel contained the indicated amounts of lysates of HEK293 transfected (+) or not transfected (-) with myc-CaM KMT pCDNA3 vector to serve as a marker for the size of CaM KMT. Page ruler prestained protein ladder (#SM1811 Fermentas): Ma. 60 μg of human lymphoblasts and mouse tissues and the indicated amounts of HEK293 lysates were separated on 12% PAA gel. The size of the CaM KMT is indicated by the blue box. Although the polyclonal antibodies were affinity purified, the figure represents many cross reactive proteins. The whole Western blot is presented as supplementary S3 Fig.