Mouse anti myc

After transfection, cells were washed with cold PBS before being solubilized in a lysis buffer containing 120 mM Tris/Hepes, pH 7,4; 150 mM NaCl, 5 mM EDTA, 3 mM KCl; 1% (v/v) Triton X-100 and protease inhibitors (Complete Roche 1697498, Meylan, France). Samples were then harvested and centrifuged at 16,000 rpm for 15 min at 4 °C. Protein expression levels were assessed after normalizing and loading equal amounts of total protein for 7.5% SDS-PAGE separation and immunoblotting with the antibodies of interest and horseradish peroxidase-conjugated secondary antibody. The primary antibodies used in this study were mouse anti–Myc (Takara, Clontech, Saint-Germain-en-Laye, France) and anti-Sar1A (Invitrogen). Proteins were visualized by enhanced chemiluminescence detection (Thermo Fisher Scientific, Les Ulis, France) according to the manufacturer′s instructions.

Proteins were separated using a Criterion^TM XT precast gel (4–12% Bis-Tris, (Biorad, Hercules, CA, USA)). After electrophoresis, proteins were transferred onto a nitrocellulose membrane using the Criterion^TM Blotter (Biorad, Hercules, CA, USA). Membranes were incubated with rabbit anti-CCP5 (1:1000, Ab118621, Abcam, Cambridge, MA, USA), mouse anti-polyglutamate (B3, 1:2000, Sigma-Aldrich), mouse anti-glutamate (GT335, 1:4000, Adipogen, San Diego, CA, USA), rabbit anti-long-chain polyglutamate (polyE, 1:4,000, Adipogen), mouse anti-myc (1:1200, Clontech, Mountain View, CA, USA) or rabbit anti-α-tubulin (EP1332Y, 1:3000, Abcam) antibodies. Immunoreactivity of proteins was visualized with Supersignal® West Pico Chemiluminescence Substrate (Thermo, Rockford, IL, USA) following incubation with HRP-labeled sheep anti-mouse (1:2000, GE Healthcare Sciences, Pittsburgh, USA) or donkey anti-rabbit IgG (1:10,000, GE Healthcare Sciences) antisera.