Ck14 tubes

For western blots, one snap-frozen lung lobe was lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 0.5% SDS, 0.1% NP-40, 0.1% Triton X-100, supplemented with PhosSTOP phosphatase inhibitor and Complete protease inhibitor cocktail (both Roche Diagnostics), using a Precellys24 homogenizer and CK14 tubes (Bertin Technologies SAS, Montigny le Bretonneux, France). Tissue debris were removed from the lysates by centrifugation at 4°C for 30 min at 10,000 rpm 10 μg of total proteins were separated by SDS-PAGE on 4–12% gradient gels (Biorad) and transferred to PVDF membranes (Immobilon-P, Millipore). For the detection of proteins, the following primary antibodies were used: goat anti-SP-C, rabbit anti-SP-A (Santa Cruz Biotechnologies, 1:500), rabbit anti-Actin (Cell Signaling Technologies, 1:2000), rabbit anti-calnexin ([44 (link)], 1:5000).

Samples were collected at the indicated time points and immediately frozen on dry ice and stored at −80 °C. Tissues were homogenized in Trizol using a Precellys 24 (Bertin) bead mill homogeniser and CK14 tubes (Bertin). Lysed and homogenized samples were then processed for RNA extraction using a Direct-zol RNA MiniPrep kit according to manufacturer’s instructions. RNA quantity and quality were analyzed by Nanodrop 2000 (ThermoFisher) and 200 ng was taken for cDNA synthesis. Reverse transcription was performed with High-Capacity RNA-to-cDNA Kit and resulting cDNA was diluted to 1 ng/µl. Q-PCR analyses were performed using PowerUp SYBR Green (Applied Biosystems): primer sequences are provided in Supplementary Table 2. Quantification of transcript was performed with StepOne v2.3 software using the 2^-ΔΔCt method using Rpl32 as an internal reference gene.