Supplemental Data

Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Generation of Galnt3-deficient mice. Schematic of the gene targeting is shown. Galnt3 exons 2 and 3 were replaced with the neomycin resistance gene by homologous recombination. MC1-TK, polyoma enhancer fragment-thymidine kinase; PGK-Neo; phosphoglycerate kinase promoter-neomycin resistance; A, AseI; B, BamHI; K, KpnI; Nh, NheI; Ns, NsiI. Not all restriction sites are shown.
• Supplemental Figure 2 - Absence of Galnt3 expression and activity in Galnt3-deficient mice. (A) Galnt3 genotyping of F2 mice by Southern blot analysis (top) and PCR genotyping (bottom). In Southern blot analysis, genomic DNA was digested with AseI and probed with the 3’ probe that detects restriction fragments (double-headed arrows) depicted in Supplemental Figure 1. (B) Quantitative RT-PCR analysis of Galnt3 gene expression in the kidney. N = 11-12 per genotype. The data are presented as mean fold change ± SEM. **, P < 0.01 (versus wild-type control mice). (C) Detection of Galnt3 by immunoblotting. A lysate of HEK293 cells transfected with mouse Galnt3 cDNA (IMAGE clone 5342768; Accession no. BC043331) was used as a positive control, C. (D) GalNAc transferase assay. The acceptor peptide based on the HIV gp120 protein was used to quantify transferase activity specific to Galnt3. EA2 is a nonselective peptide used as a control. The data are presented as mean activity ± SEM. **, P < 0.01 (versus wild-type control mice) based on two replicates from each of two mice per genotype. Bars: Wild-type, white; heterozygote, gray; and homozygote, black.
• Supplemental Table 1 - Primers and assays used for quantitative RT-PCR.
• Supplemental Table 2 - Serum biochemical profiles of tumoral calcinosis patients and mouse models.