Bolt 12 bis tris plus

To label newly synthesized mitochondrial DNA-encoded proteins, cells were seeded into a six-well dish at 80–90% confluency. First, two washing steps of 5 min each in methionine/cysteine-free DMEM were performed. Subsequently, cells were incubated with fresh methionine/cysteine-free DMEM supplemented with Glutamax 100X (Gibco), sodium pyruvate 100X (Gibco), 10% dialysed FBS and 100 µg ml⁻¹ emetine (Sigma-Aldrich) for 20 min at 37 °C. Labelling was performed with the addition of 166,7 µCi ml⁻¹ of EasyTag EXPRESS [³⁵S] protein labelling mix (methionine and cysteine) (Perkin Elmer) for 30 min at 37 °C. Following labelling, cells were washed with 1 ml of PBS three times and the final pellets were collected by centrifugation. Cells were lysed in 1× PBS-PIC with the addition of 50 units of benzonase (Life Technologies) with incubation on ice for 20 min, followed by the addition of SDS to 1% final concentration. and further incubation on ice for 30 min. After cell lysis, 30 µg total protein was separated on Bolt 12% Bis-Tris Plus (Invitrogen) SDS–PAGE gels. Gels were then incubated in Imperial Protein Stain (Thermo Fisher) for 1 h and with fixing solution (20% methanol, 7% acetic acid and 3% glycerol) for 1 h. Next, gels were vacuum-dried at 65 °C for 2 h. The resultant gel was exposed to storage phosphor screens and visualized with Typhoon FLA 7000 Phosphorimager.

Cell pellets were lysed in lysis buffer (20mM Tris (pH8), 150mM NaCl, 0.5% NP-40, 1mM EDTA) supplemented with protease inhibitors (cOmplete EDTA-free protease inhibitor cocktail; Roche) and phosphatase inhibitor cocktails 2 and 3 (P5726 and P0044; Sigma). Lysates were heated at 70°C for 10min in NuPAGE LDS sample buffer. Proteins were separated by SDS-PAGE on a Bolt 12% Bis-Tris Plus or a NuPAGE 4–12% Bis-Tris gel (Invitrogen) and transferred to polyvinylidene difluoride (PVDF) membrane in NuPAGE transfer buffer plus 7.5% methanol. The membranes were blocked in Tris-buffered saline plus 0.1% Tween 20 (TBST) plus 5% milk powder then incubated with primary antibodies: pan-14-3–3 antibody (8312S, Cell Signaling Technology), V5 antibody (R96025, Invitrogen), 14-3-3γ (Cell Signaling Technology, 5522), FOXO3a (Cell Signaling Technology 12,829), FLAG (Sigma, F1804), HSP90 (Cell Signaling Technology, 4877S), pan-AKT (Cell Signaling Technology, 2920S), AKT-pSer473 (Cell Signaling Technology, 4060S), FOXO3a-pSer253 (Cell Signaling Technology, 9466S), beta-actin (Sigma Aldrich, A5441), YAP (Cell Signaling Technology, 12395S), p53 (Santa Cruz, sc-126). Membranes were then incubated with horseradish peroxidase (HRP) conjugated secondary antibodies and developed using Amersham ECL Western Blotting Detection Reagents.

Protein samples were mixed with an equal volume of 2× SDS-PAGE sample buffer and heated to 100°C for 10 min prior to electrophoresis on Bolt 12% Bis-Tris Plus gels (Invitrogen). Proteins were visualized using SimplyBlue SafeStain (Invitrogen).