Revert total protein stain solution

Cells and brain tissue were lysed in ice-cold RIPA 1× (cat no 20-188, Merck), supplemented with protease inhibitors. Samples were spin at 12,000 × g for 15 min at 4 °C. The supernatant was collected, and protein concentration was determined via BCA Protein Assay (cat no 23227, ThermoFisher Scientific) following manufacturer’s instructions. For Western blot, 5–30 μg protein were separated on pre-casted 4-15% Mini-PROTEAN TGX Gels (cat no 4561086 and 4561084, Bio-Rad) and transferred onto nitrocellulose membranes (Trans-Blot Turbo Midi NC Transfer, cat no 170-4159, BioRad) using a semi-dry transfer protocol. Before blocking, the total amount of protein per lane was determined using REVERT Total Protein Stain Solution (cat no 926-11011, Li-Cor) and used for normalization. Blocking was performed in 3% BSA in TBST (0.02 M Tris base, 0.15 M NaCl, 0.05% Tween-20 in milliQ water, pH 7.2–7.4) for 1 h at RT and incubated overnight at 4 °C with the primary antibodies diluted in blocking buffer. Membranes were then washed 3× with TBST for 5 min at RT and incubated with secondary antibodies (IRDye, 1:15000 in blocking buffer, Li-Cor). After 3 washes with TBST, membranes were imaged with the Li-Cor Odyssey system. Uncropped blots are provided with this paper as Source Data.

Protein extracts were performed by lysing cells in NP-40 lysis buffer (1% NP-40, 20 mM Tris pH 8, 137 mM NaCl, 10% glycerol, 2 mM EDTA), containing protease and phosphatase inhibitor (Thermo Scientific). Protein extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes (BIO-RAD). In some experiments, equal protein loading was controlled by staining the membrane after transfer using REVERT total protein stain solution (LI-COR). Membranes were blocked in TBS 5% milk and probed with primary antibodies overnight, washed 4 × 5 min in TBS + 0.1%Tween before incubating with secondary antibodies conjugated to AlexaFluor680 or IRDye800 for 1 h, washed and read on Odyssey CLx imaging system (LI-COR). Proteins were quantified using ImageStudiolite software. When Revert staining (LI-COR) was used, Revert signal from a whole protein lane was considered for normalization. The antibodies are listed in Supplementary Data 5. The original uncropped images of all western blots are shown in Supplementary Fig. 7.

Proteins were purified, separated and blotted as described with modifications (Frenk et al., 2014 (link)): aged MEP cells were re-suspended in Laemmli buffer containing 100 mM DTT and separated on 15% acrylamide gels, and Odyssey blocking buffer PBS (LI-COR 927 – 40000) was used for antibody hybridisations. Membranes were probed with antibodies listed in Table S7 at given dilutions before imaging on a LI-COR Odyssey 9120 imager. Total protein detection was performed by staining membranes with REVERT Total Protein Stain solution (LiCor 926 – 11010) after antibody detection.