Odyssey 2.1 system

Briefly, cells were washed twice in ice-cold PBS and lysed in RIPA (Sigma-Aldrich R02780) buffer supplemented with Protease (Roche 11697498001) and phosphatase inhibitors (Sigma-Aldrich 93482) for 10 minutes in ice with intermittent vortexing. Samples were cold-centrifuged at maximum speed for 15 minutes and supernatant transferred to a clean cold eppendorf. The protein concentration of each sample was determined using a bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Waltham, MA, USA, 23227). Equal amounts of lysates were subject to immunoblotting on SDS-PAGE. Proteins were transferred to a Biotrace NT membrane (VWR, Radnor, PA, USA, PN66485) and incubated with primary antibodies. Proteins were visualized using donkey anti-mouse, and anti-rabbit secondary antibodies conjugated to the IRDyes, IR680-LT (926-68022) or IR800 (926-32213) (LI-COR Biosciences, Lincoln, NE, United States) and the LI-COR Odyssey 2.1 system. 16-bit images were analysed and quantified using the Odyssey analysis software.

Whole-cell extracts were prepared by direct addition of hot Laemmli buffer and incubation at 110 °C for 10 min with intermittent vortexing. Following bicinchoninic acid assay (Thermo Scientific), equivalent amounts of proteins were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to BiotraceNT membrane (VWR) for incubation with primary antibodies. To accentuate band shifts caused by phosphorylation, samples were run on 6% polyacrylamide gels with 20 µM Phos-Tag Acrylamide (NARD institute, AAL-107) and 40 µM MnCl_2. Proteins were visualised using donkey anti-mouse, anti-rabbit, or anti-sheep secondary antibodies conjugated to the IRDyes IR680-LT, or IR800 (LI-COR), and a LI-COR Odyssey 2.1 system, with 16-bit images quantified in ImageStudio.