Western blot was performed on samples from untreated and fibrotic wt and KCa3.1−/− mice (12 weeks CCl4 exposure) for Proliferating Cell Nuclear Antigen (PCNA; Santa Cruz-Biotechnology, Santa Cruz, USA). Proto-oncogene tyrosine-protein kinase Src (c-Src) activity was analysed by phosphorylation either at Thr418 or at Thr530 (both Santa Cruz-Biotechnology, Santa Cruz, USA). Activation of c-Jun N-terminal kinases 1 and 2 (pJNK1&2; Life technologies, Darmstadt, Germany) and total JNK (Santa Cruz-Biotechnology, Santa Cruz, USA) was assessed by their phosphorylation at Thr183, respectively Tyr185. GAPDH served as endogenous control.
Snap frozen liver samples were processed as previously described using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)20 (link)21 (link)26 (link). Ponceau S staining was used to confirm equal protein loading. Membranes were blocked and incubated with respective antibodies. Thereafter, membranes were incubated with the corresponding secondary peroxidase-coupled antibodies (Santa Cruz-Biotechnology, Santa Cruz, USA).
After enhanced chemiluminescence (ECL; Amersham, Bucks, UK), digital detection was evaluated using Chemi-Smart (PeqLab Biotechnologies, Erlangen, Germany). Data are expressed as means ± standard error of the mean (SEM) with values of controls normalized to 100%.
Snap frozen liver samples were processed as previously described using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)20 (link)21 (link)26 (link). Ponceau S staining was used to confirm equal protein loading. Membranes were blocked and incubated with respective antibodies. Thereafter, membranes were incubated with the corresponding secondary peroxidase-coupled antibodies (Santa Cruz-Biotechnology, Santa Cruz, USA).
After enhanced chemiluminescence (ECL; Amersham, Bucks, UK), digital detection was evaluated using Chemi-Smart (PeqLab Biotechnologies, Erlangen, Germany). Data are expressed as means ± standard error of the mean (SEM) with values of controls normalized to 100%.
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