Lumi lightplus western blotting substrate

Western blotting was done as previously described [41 (link)]. In brief, cells were lysed in lysis buffer (20 mM Hepes, 1 mM EGTA, 5 mM MgCl2, 100 mM NaCl, 4 mg/mL Decyl-beta-d-maltopyranoside, 10 % glycerol and protease inhibitor cocktail). Cell lysates were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. After membranes were blocked with 5 % BSA for 1 h, they were probed with indicated primary antibodies (8 μg/mL) overnight at 4 °C, followed by incubation with the HRP-conjugated secondary antibodies for 1 h at room temperature, and finally visualized with the Lumi-Light^PLUS Western blotting Substrate (Roche).

Cell extracts were run on 5–20% gradient SDS–polyacrylamide gel electrophoresis (PAGE; ATTO) or 7.5% SDS–PAGE and then transferred to Hybond ECL membranes (GE Healthcare) followed by western blot analysis with the indicated antibodies. Detection was conducted with Lumi-Light PLUS Western Blotting Substrate (Roche) or SuperSignal West Pico Chemiluminescent Substrate (Thermo) and images were obtained with LAS3000 (Fujifilm).

Kc167 cells were collected, washed in PBS and incubated for 30 min in IP buffer (150 mM NaCl, 0.5% NP40, 50 mM Tris-HCl, pH8.0, 1mM EGTA) supplemented with protease inhibitor cocktail (Roche). The extracts were cleared by centrifugation at 13.000g for 15 min at 4°C and subjected to SDS-PAGE (50 μg of proteins par lane) or immunoprecipitation (1 mg per point). For immunoprecipitation, proteins were preadsorbed with 100 μl of sepharose beads slurry for 1h at 4°C before being incubated with 20 μl of anti-GFP (Chromotek), anti-V5 (Sigma-Aldrich) or anti-HA (Covance) antibody coupled to sepharose beads, or with 10 μl of rabbit anti-MLF [19 (link)] or rabbit IgG (SantaCruz) in the presence of 20 μl of protein A sepharose beads (Sigma), for 4h at 4°C. The beads were spun down and washed in IP buffer and immunoprecipitated proteins were processed for SDS-PAGE and Western Blot analyses. Western blots were performed using standard techniques and the blots were developed by photoluminescence procedure using Lumi-Light^PLUS Western Blotting Substrate (Roche) and Amersham Hyperfilm^TM ECL (GE Healthcare) or Chemidoc Touch Imaging System (BioRad). The following antibodies were used for Western blots: anti-V5 (Invitrogen), anti-HA (BioLegend), anti-GFP, anti-tubulin (Sigma-Aldrich), anti-Renilla luciferase (MBL), and anti-MLF [19 (link)].