CADM1 Shedding and Lung Protein Analysis

Cells and mouse lungs were lysed in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100 and 1 mM phenylmethylsulfonyl fluoride and were subjected to Western blot analyses as described in our previous report (Koma et al., 2008 (link)). The recovered bronchoalveolar lavage fluid was directly separated on SDS-PAGE gels. Two kinds of anti-CADM1 antibodies were used, which we previously generated; rabbit polyclonal antibody against the C-terminal peptide (eggqnnseekkeyfi) to detect full-length CADM1 and αCTF (Mimae et al., 2014 (link)), and chicken monoclonal antibody against the ectodomain (3E1) to detect the N-terminal fragment (NTF) released from the cell by CADM1 shedding (Furuno et al., 2005 (link); Nagara et al., 2012 (link)). Other primary antibodies used in this study targeted ADAM10 (rabbit polyclonal; Millipore, Billerica, MA, USA), αSMA (mouse monoclonal clone 1A4; Dako, Santa Clara, CA, USA), E-cadherin (clone 36; BD Bioscience, San Jose, CA, USA), pan-cytokeratin (mouse monoclonal AE1/AE3; Dako), and β-actin (Medical & Biological Laboratories). Peroxidase-conjugated secondary antibodies were purchased from Amersham (Buckinghamshire, England). Immunoreactive band intensities were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA), as described previously (Mimae et al., 2012 (link)).