For cellular protein lysates, cells were scraped on ice using cold Ripa lysis buffer (150 nM NaCl, 50 mM Tris–HCl pH 8, 1% Igepal, 0.5% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (CompleteTM, Roche), 1 mM Na3VO4 (Sigma), 100 mM NaF (Sigma), and 1 mM DTT (Sigma).
Proteins were separated in 4–20% SDS–PAGE (Criterion Precast Gel, Bio‐Rad) and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% dried milk in TBS‐0,1% Tween 20 or in Odyssey Blocking Buffer (LI‐COR, Biosciences) and incubated at 4°C overnight with primary antibodies. The list of primary antibodies is provided in Appendix TableS5 .
Membranes were washed in TBS‐0,1% Tween 20 and incubated 1 h at RT with IR‐conjugated (AlexaFluor680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, LI‐COR) or with the appropriate horseradish peroxidase‐conjugated secondary antibodies (GE Healthcare) for ECL detection (Clarity Western ECL Substrate, Bio‐Rad). Band quantification was performed using the Odyssey v1.2 software (LI‐COR) or the QuantiONE software (Bio‐Rad Laboratories). The Re‐Blot Plus Strong Solution (Millipore) was used to strip the membranes, when reblotting was needed.
Proteins were separated in 4–20% SDS–PAGE (Criterion Precast Gel, Bio‐Rad) and transferred to nitrocellulose membranes (GE Healthcare). Membranes were blocked with 5% dried milk in TBS‐0,1% Tween 20 or in Odyssey Blocking Buffer (LI‐COR, Biosciences) and incubated at 4°C overnight with primary antibodies. The list of primary antibodies is provided in Appendix Table
Membranes were washed in TBS‐0,1% Tween 20 and incubated 1 h at RT with IR‐conjugated (AlexaFluor680, Invitrogen or IRDye 800, Rockland) secondary antibodies for infrared detection (Odyssey Infrared Detection System, LI‐COR) or with the appropriate horseradish peroxidase‐conjugated secondary antibodies (GE Healthcare) for ECL detection (Clarity Western ECL Substrate, Bio‐Rad). Band quantification was performed using the Odyssey v1.2 software (LI‐COR) or the QuantiONE software (Bio‐Rad Laboratories). The Re‐Blot Plus Strong Solution (Millipore) was used to strip the membranes, when reblotting was needed.