Immunoblotting Analysis of Sec62 Protein

Cells (1 × 10⁶) were lysed in a lysis buffer [distilled water, 10 mM NaCl, 10 mM Tris(hydroxymethyl)aminomethane, 3 mM MgCl₂, and 5% NP-40], and the proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by blotting and subsequent immunostaining. Canine pancreatic rough microsomes (RM) were analyzed in parallel and allowed the identification of the Sec62 protein in the human cell extracts [13 (link),14 (link),17 (link),24 (link)]. We used the above-described affinity-purified polyclonal rabbit antipeptide antibody directed against the C terminus of human Sec62 (at a dilution of 1:500) and a monoclonal mouse antibody directed against the N terminus of human β-actin (at a dilution of 1:10,000) (Sigma-Aldrich, St. Louis, MO, USA). The secondary antibodies used were ECL Plex goat anti-rabbit Cy5 and anti-mouse Cy3 conjugates (GE Healthcare, Munich, Germany) (at dilutions of 1:1000 and 1:10,000, respectively). The blots were imaged using the Typhoon-Trio system and Image Quant TL software (version 7.0; GE Healthcare). The Sec62 and β-actin levels were quantified (in arbitrary signal units), and the former was normalized against the latter.