Western lightning plus ecl enhanced chemiluminescence

To detect I-SceI expression, cells were collected at the time of FACS analysis (48 h after addition of doxycycline). Cells were lysed for 20 min on ice in TEGN buffer (10 mM Tris (pH 8), 1 mM EDTA, 10% glycerol, 0.5% Nonidet P-40, 400 mM NaCl) with 1 mM DTT in the presence of protease inhibitors (Roche). Lysates were cleared by centrifugation at 10,000g for 10 min. Total protein concentration was determined, and 30 μg protein was loaded onto 10% SDS-polyacrylamide gel electrophoresis gels and transferred to nitrocellulose (Bio-Rad). Membranes were blocked for 1 h in 5% milk prepared in PBS with 0.1% Tween-20 and probed overnight at 4 °C with anti-HA (1:1,000 in 5% milk, Covance clone 16B12, MMS-101) or anti-tubulin antibodies (1:10,000 in 5% milk, Sigma clone DM1A, T9026). After washing, membranes were incubated with anti-mouse horseradish peroxidase-linked secondary antibody (1:10,000 in 5% milk) for 1 h. Proteins were visualized with Western Lightning Plus-ECL Enhanced Chemiluminescence (Perkin Elmer). Uncropped images of Western blots are shown in Supplementary Fig. 5.

Protein samples were obtained from cell lysates using lysis buffer (#9803; Cell Signaling) containing PhosSTOP (#04-906-845-001; Merck) and Complete Protease Inhibitor Cocktail (#11-697-498-001; Merck). Protein samples were reduced with β-mercaptoethanol or dithiothreitol and heated at 95°C with shaking. Samples were loaded on 10% SDS–Page and run at 120 V. Protein samples were transferred onto polyvinylidene fluoride (PVDF) membranes previously activated with methanol. After blocking for 1 h with 4% BSA (#A7906; Merck), the membrane was incubated overnight with primary antibodies diluted in 4% BSA. GAPDH was used for normalisation. Horseradish peroxidase-conjugated antibodies (Jackson Immuno Research) were used as secondary antibodies with incubation for 1 h. Western Lightning Plus-ECL Enhanced Chemiluminescence (NEL105001EA; Perkin Elmer) was used as substrate to develop the membrane in a colorimetric or fluorescence detection. Image SXM or Fusion FX7Edge software were used to quantify protein expression. For multiple stainings, the membranes were re-blotted after stripping with Re-Blot Plus Strong Solution (#2504; Merck).