Monoclonal mouse anti flag m2 f3165

Cells were lysed in cold buffer containing 150 mM NaCl, 20 mM HEPES pH 7.5, 10 mM EDTA and 1% Triton X-100, and supplemented with protease inhibitors cocktail (Roche) and 20 mM NEM. Protein concentration was determined by standard BCA assay (Pierce). Samples with equal protein concentration were mixed with non-reducing Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol and 0.01% bromophenol blue) and heated for 5 min at 95°C. For reducing SDS-PAGE, samples were supplemented with 100 mM DTT. Protein samples separated by either reducing or non-reducing SDS-PAGE were then transferred to Protran nitrocellulose membrane (Merck) and probed with the following antibodies: mouse pan-actin (clone C4) and mouse GAPDH (AB9484, Abcam) from Sigma Aldrich, monoclonal mouse anti-FLAG M2 (F3165, Sigma Aldrich), monoclonal mouse anti-KDEL (ADI-SPA-827, Enzo life Sciences), Selenoprotein N (A-11) from Santa Cruz Biotechnology, mouse OXPHOS (ab110413) from Abcam and rabbit ERO1.⁴⁹ (link)

Anisomycin, the polyclonal rabbit anti-B4GALNT2 antibody (HPA015721), the monoclonal mouse anti-GFP (G1546), the monoclonal mouse anti-Flag M2 (F3165), the polyclonal rabbit anti-TMEM165, and the mouse anti-β-actin antibodies were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Anti-Sd^a (KM694) antibody was a kind gift from Dr. Taeko Dohi and Dr. Akiko Furuya (Biologics Research Laboratories, Research Division, Kyowa Hakko Kirin Co., Tokyo, Japan). The polyclonal goat anti-mouse horseradish peroxidase-conjugated IgG was purchased from Agilent Technologies (Santa Clara, CA, USA). The polyclonal goat anti-mouse horseradish peroxidase-conjugated IgM was purchased from Fisher Scientific (Illkirch, France). The polyclonal goat anti-rabbit horseradish peroxidase-conjugated IgG was purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). The secondary antibodies Alexa fluor^® 488 anti-rabbit and Alexa fluor^® 568 anti-rabbit were from Life Technologies (Fisher Scientific, Illkirch Graffenstaden, France). The polyclonal rabbit anti-Calnexin was purchased from Enzo life technologies (Villeurbanne, France). The GFP expression vector pFx was a kind gift of Dr. Jack Röhrer (University of Zurich), the expression vectors pEGFP-N1 and pmCherry-N1 were from Clontech, whereas p3×FLAG-CMVTM-10 was from Sigma.

Prior to separation by 10% SDS-PAGE, TSP- and IF-derived proteins were mixed with reducing 4× Laemmli buffer and incubated for 5 min at 95 °C. In experiments where nonreducing conditions were required, β-mercaptoethanol was excluded from 4× Laemmli buffer. Fractionated proteins were used for immunoblotting or Coomassie Brilliant Blue R-250 staining. Protein- and tag-specific antibodies were used for immunoblot analysis (polyclonal goat anti-BChE N-15; Santa Cruz Biotechnology, Inc® (Heidelberg, Germany) sc-46803, diluted 1 : 300 and monoclonal mouse anti-FLAG M2, F3165; Sigma Aldrich®, diluted 1 : 10000). Detection was performed using HRP-conjugated secondary antibodies (anti-goat IgG-peroxidase antibody A5420, and anti-mouse IgG-peroxidase antibody A9044, both diluted 1 : 10000 from Sigma Aldrich®). Clarity™ Western ECL from Bio-Rad was used as a substrate.