Mineralization of vibrissae dermal sheath in mice placed on experimental “acceleration diet” at the age of 4 weeks and sacrificed at 5 months of age.

<p>Note the absence of mineralization in the C57BL/6J mice and extensive mineralization in <i>Abcc6^−/−</i> mice when examined by hematoxylin-eosin (HE) and by Alizarin red (AR) stains. Note ectopic mineralization in the four mouse strains with the A allele of the <i>Abcc6</i> gene (arrows).</p

Analysis of <i>ABCC6</i> mRNA levels in the liver.

<p>(A) Relative mRNA levels in the mice with the A allele (KK/HlJ, DBA/2J, C3H/HeJ and 129S1/SvImJ), in comparison to the mouse with the G allele, C57BL/6J. Note that the mRNA level is essentially undetectable in <i>Abcc6^−/−</i> mice; (n = 4–5; *p<0.05 in comparison to other groups; Student’s t-test). (B) Separation of the G and A alleles by fine resolution gel electrophoresis, represented by 144 and 139 bands, respectively.</p

Western blot analysis of ABCC6 protein isolated from the liver of mice.

<p>(A) The blot was stained with a mouse anti-ABCC6 antibody S20 (upper panel). The same blot was rehybridized with an antibody for sodium potassium ATPase as a control of loading of plasma membrane proteins (lower panel). (B) The relative concentration of the protein, as determined from the Western blots in A by scanning densitometry, normalizing the ABCC6 protein levels by those with sodium potassium ATPase (n = 4–5; p<0.05 in comparison to other groups; Student’s t-test). (C) Lack of conservation of the arginine (R) in position p.621 of ABCC6 in different vertebrates.</p

Immunofluorescence staining of Abcc6 in the liver of mice.

<p>Note positive staining associated with the plasma membranes in C57BL/6J mice (arrowheads) and complete absence of the staining in <i>Abcc6^−/−</i> mice. A low, yet detectable level of immunofluorescence was noted in the four other strains which harbor the A allele of the <i>Abcc6</i> gene.</p

Vibrissae dermal sheath mineralization in different mouse strains examined in this study.

<p>The table shows the frequency and severity of vibrissae dermal sheath mineralization in different strains: −, not present; +, mild; ++++, severe. Mice were placed on either normal diet or acceleration diet at 4 weeks of age and tissues were collected for histopathology. The frequency values represent the percent of mice affected by vibrissae dermal sheath mineralization, as examined by Hematoxylin-Eosin stain on one section.</p