Anti mouse cy3 conjugates

2 × 10⁵ cells were lysed in a lysis buffer (aqua dest. + 10 mM NaCl/10 mM Tris (hydroxymethyl)-aminomethan/3 mM MgCl₂/5 % NP-40) and proteins were resolved by SDS-PAGE and identified by immunoblotting. We used an affinity-purified polyclonal rabbit anti-peptide antibody directed against the C terminus of human SEC62 (self-made), a polyclonal rabbit antibody directed against the C terminus of human SOX2 (abcam pic, Cambridge, UK) and a monoclonal mouse antibody directed against the N terminus of human b-actin (Sigma-Aldrich Co., St. Louis, MO, USA). The secondary antibodies used were ECL Plex goat anti-rabbit Cy5 or anti-mouse Cy3 conjugates (GE Healthcare, Munich, Germany). Blots were imaged using the Typhoon-Trio system and Image Quant TL software 7.0 (GE Healthcare, Munich, Germany). The SEC62, SOX2 and β-actin levels were quantified and normalized against β-actin.

2 × 10⁵ HeLa cells were lysed in a lysis buffer (aqua dest. + 10 mM NaCl/10 mM Tris(hydroxymethyl)-aminomethan/3 mM MgCl₂/5 % NP-40) and proteins were resolved by SDS-PAGE and identified by immunoblotting. Antibodies used were the previously described anti-human Sec62, monoclonal anti-human β-actin (Sigma-Aldrich Co., St. Louis, MO, USA), anti-human E-cadherin Clone 24E10 (Cell signaling Technology, Cambridge, UK), anti-human vimentin Clone V9 (Dako Denmark A/S, Glostrup, Denmark) and anti-human GAPDH (sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA) antibody. Secondary antibodies used were ECL Plex goat anti-rabbit Cy5 or anti-mouse Cy3 conjugates (GE Healthcare, Munich, Germany). Blots were imaged with the Typhoon-Trio system and the Image Quant TL software 7.0 (GE Healthcare, Munich, Germany). Sec62, vimentin, and β-actin levels were quantified and normalized to GAPDH.

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.