The total protein concentration of PMN lysate, BALF and whole lungs in RIPA lysis buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% (w/v) deoxycholic acid, 0.1% (w/v) SDS, 0.5% (v/v) Nonidet P-40, pH 8.0) has been determined by Pierce™ BCA Protein AssayKit. BALF and lysates were separated on 12% SDS-PAGE under denaturating conditions and 10–15 µg per lane were transferred to a polyvinylidene difluoride (PVDF) membrane. Free sites on the membrane were blocked by incubation with 1× Roti®block (Carl Roth). Membranes were then incubated with goat anti-mouse PR3 polyclonal antibody (1:1000), rabbit anti-mNE (1:1000, ab68672, Abcam), rabbit anti-mCatG (1:1000, ab197354, Abcam) and rabbit anti-alpha 1 antitrypsin (1:1000, ab133642, Abcam) in 1× Roti®block at 4 °C overnight. Detection of bound primary antibodies was performed by ECL after mouse α-goat IgG (1:20000, Merck), goat α-rabbit (1:40000, SigmaAldrich), goat α-mouse IgG + IgM (1:40000, Jackson ImmunoResearch)) incubation.
Mouse elastase concentrations in whole cell lysates and IL-6 levels in BAL fluid were quantified by ELISA (PicoKineTM Elisa, (EK1445), Multi Analyte ELISArray Kit (MEM-004A)) and conducted as described in the manufacturer’s recommendations.
Mouse elastase concentrations in whole cell lysates and IL-6 levels in BAL fluid were quantified by ELISA (PicoKineTM Elisa, (EK1445), Multi Analyte ELISArray Kit (MEM-004A)) and conducted as described in the manufacturer’s recommendations.