Actin isoform expression after liver injury.

<p>In (A–C), stellate cells were isolated after carbon tetrachloride (CCl₄) induced liver injury as in Methods and plated on glass coverslips. Twenty-four hours later, smooth muscle α actin (<i>Acta2</i>) (A, Texas red) and nonmuscle β-actin (B, FITC) were detected by immunocytochemistry as in Methods. In (C and D) are shown overlays, revealing co-localization of actins (C: bar = 10 microns; D: bar = 5 microns). Identical results were obtained with cells after either form of liver injury, and images are representative of over 20 others. In (E), stellate cells were isolated from normal livers or 8 days after bile duct ligation or 10 doses of carbon tetrachloride and immediately subjected to immunoblotting as in Methods. Representative immunoblots shown depict duplicate, identical, samples probed for each <i>Acta2</i> and anti-cytoplasmic β actin (7.5 µg total protein). In (F), specific bands were scanned, quantitated and expressed graphically (n = 4 for each model of injury, *p<0.001 compared to normal). In (G), stellate cells from normal or injured livers were immediately lysed and equal amounts (40 µg) of cellular proteins were subjected to 2-D gel electrophoresis as in Methods. Notably, we also made a theoretical estimation of isoactin PIs by in silico analysis of each actin isoform (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077166#pone.0077166-Bjellqvist1" target="_blank">[67]</a>(<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077166#pone.0077166.s001" target="_blank">Figure S1</a></b>)). Representative examples (of greater than 20 separate experiments) reveal specific actin isoforms, and after injury (bile duct ligation), new expression of an α isoform (two-D gels are shown in the standard international format with pI ranging from acidic to basic, left to right). In (H), a representative immunoblot of similarly prepared protein samples after 2-D gel electrophoresis is shown (200 µg total protein each). As described in Methods, nitrocellulose membranes were probed sequentially with anti-cytoplasmic β-actin then anti-<i>Acta2</i> (using the same ECL detection method each time, thus accounting for repeat detection of the β-actin band). Abbreviations: <i>Acta2</i> - smooth muscle α actin; BDL - bile duct ligation; CCl₄ - carbon tetrachloride.