Antibody to gapdh

Detection of Dnaic1 protein by Western blotting was performed using a mouse monoclonal antibody generated against a synthetic peptide from the human DNAI1 protein ^{18 (link)} using standard procedures. For analysis of mouse tracheal epithelial cells, total cell lysates were prepared in Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Ciliary axonemes were isolated from cultured mouse tracheal epithelial cells as previously described ^{32 (link)} and prepared in gel-loading buffer. For studies of protein turnover, both the soluble (cytoplasmic) and pellet (axonemal) fractions from individual cultures were analyzed. Protein samples were fractionated on 4 to 12% Bis-Tris gradient gels (Invitrogen, Carlsbad, CA), transferred to nitrocellulose membranes, and probed using Amersham ECL Plus reagents (GE Healthcare, Buckinghamshire, UK), all according to manufacturer's instructions. For normalization of loading, Western blots of cytoplasmic extracts were reprobed with an antibody to GAPDH (Sigma, St. Louis, MO), and axonemal pellets were reprobed with mouse anti-acetylated α-tubulin (Invitrogen). Quantification of signals was performed on an Odyssey Imaging System (Licor, Lincoln, NB).

The cells were lysed with RIPA lysis buffer (Millipore, Billerica, MA) containing a complete protease inhibitor tablet (Sigma). The protein concentration of tumor extracts was determined using BCA Protein Assay Kit (Pierce). Lysates were cleared by centrifugation at 14,000g for 20 min. The supernatant fractions were used for western blot. Protein extracts were resolved by 15% SDS-PAGE and probed with NUPR1(p8) Polyclonal Antibody (Cat #PA1–4177, ThermoFisher Scientific). Antibody to GAPDH (Millipore) was used as loading controls. The Ab binding was revealed using an HRP-conjugated anti rabbit IgG (1:3000, Cell Signaling), or anti-mouse IgG (1:3000, Amersham Pharmacia Biotech, Piscataway, New Jersey, USA). Antibody complexes were detected by SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and ChemiDoc Imaging System (Bio-Rad, Hercules, CA).