Nir fluorescence technology

For preparation of total cell lysates, cells were collected by trypsinization and centrifuged at 250 × g for 5 min, at 4 °C. Supernatant was removed, and the pellet was washed once with ice-cold PBS and then centrifuged again as described before. Cell pellet was resuspended in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, and 1% w/v Triton X-100 supplemented with 1× protease inhibitor cocktail (Sigma), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM NaF and 1 mM Na3VO4 for 30 min, at 4 °C. After centrifugation at 12,000 × g for 30 min, at 4 °C, the supernatant was collected as total cell lysate. The protein concentration was determined using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA). Blots were developed by using the NIR Fluorescence technology (LI-COR GmbH, Germany) or the ECL enhanced chemiluminescence procedure (GE Healthcare, Piscataway, NJ), the latter case indicated in each figure capture. Images were acquired and quantified by using an Odyssey CLx Infrared Imaging system (LI-COR GmbH, Germany) or by using a Chemidoc XRS video densitometer (Bio-Rad, Hercules, CA), respectively. Original uncropped images of western blots used in this study can be found in Supplementary Figs. 10 and 11.

Whole cell extracts for Western blotting analyses were prepared as previously described [12 (link)]. Membranes were developed by using the ECL enhanced chemiluminescence procedure (GE Healthcare, Piscataway, NJ) or by using the NIR Fluorescence technology (LI-COR GmbH, Germany), as indicated in each figure capture. Images were acquired and quantified by using a Chemidoc XRS video densitometer (Bio-Rad, Hercules, CA) or an Odyssey CLx Infrared Imaging system (LI-COR GmbH, Germany). A list of antibodies used is given in the Supplementary Information.