Cathepsin H function in production of lung surfactant proteins.

<p>(<b>A</b>) mRNA expression of surfactant proteins A1, B, and C measured by quantitative ‘real-time’ RT-PCR in lungs of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice (n = 5 per group). (<b>B</b>) Detection of surfactant protein B (SP-B) in lung tissue lysates (<b>C</b>) Western blot detection of SP-B in broncho-alveolar lavage (BAL) of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice of 2 genetic backgrounds (129P2/OlaHsd and C57BL/6N). The lysosomal membrane associated protein 2a (Lamp 2a) is present at the limiting membrane of lamellar bodies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026247#pone.0026247-Wasano1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0026247#pone.0026247-Albrecht1" target="_blank">[28]</a> and serves as loading control independent of the surfactant proteins. (<b>D</b>–<b>F</b>) Surface activity of BAL fluid measured by pulsating bubble surfactometry (n = 6–10).</p

Targeted disruption of the cathepsin H (Ctsh) gene.

<p>(<b>A</b>) Scheme for the targeted disruption of mouse Ctsh gene by homologous recombination. Exons are indicated by number. (<b>B</b>) Southern blot analysis of SacI-digested genomic DNA from mouse liver by the 5′ external probe denoted in panel A. Expected fragment sizes are 5.5 kb for wild-type and 6.8 kb for mutant <i>Ctsh</i> alleles. (<b>C</b>) Northern blots from liver and kidney samples of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice. The Ctsh 5′ probe detects the genuine 1.6 kb mouse Ctsh transcript in the <i>Ctsh^+/+</i> samples. *Denotes an enlarged transcript in <i>Ctsh</i>^−/− consisting of Ctsh exons 1–5 plus lacZ reporter.</p

Cathepsin expression and gross phenotype of Ctsh-deficient mice.

<p>(<b>A</b>) Western blots for Ctsh detection in lungs and liver of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice. (<b>B</b>) Detection of “acidic” aminopeptidase activity at pH 6.0 in lungs, livers and kidneys of f <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice (n = 3). (<b>C</b>) Observed and expected genotype frequencies of litters from <i>Ctsh</i>^+/− x <i>Ctsh</i>^+/− matings. (<b>D</b>) Weight gain of female littermates from heterozygous matings (n = 5 per genotype). (<b>E</b>) mRNA expression of cathepsin C (Ctsc) and cathepsin E (Ctse) measured by quantitative ‘real-time’ RT-PCR in lungs of <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− mice (n = 5 per group). (<b>F</b>) Cathepsin C (Ctsc) and cathepsin E (Ctse) detected by Western blotting in <i>Ctsh^+/+</i> and <i>Ctsh</i>^−/− lung lysates.</p