0.45 μm pore size nitrocellulose membrane

Samples were mixed with 4X sample buffer (25 M Tris (pH 6.8), 20% SDS, Glycerol, 100 mg Bromo Blue) containing 100 mM dithiothreitol (DTT), boiled for 5 min and centrifuged at 10000 g for 5 mins. Protein was electrophoresed at 270 V for 25 min on 4–20% polyacrylamide gels (BioRad; USA) and transferred on to 0.45 μm (pore-size) nitrocellulose membranes (BioRad; USA). Membranes were blocked in tris-buffered saline with 0.05% Tween20 (TBS-T) containing 5% low-fat milk powder (Diploma, Australia) for 1 h at room temperature, incubated with primary antibodies overnight at 4 °C, and incubated with secondary antibodies for 2 h at room temp. All antibodies (BD Biosciences anti- α-syn, catalogue number: 610786, dilution 1:5000; Novus Biological anti-p62/SQSTM1, catalogue number: 3868, dilution 1:2000; Cell Signalling Technology anti-LC3B(D11)-XP, catalogue number: H00008878-MO1, dilution 1:2000) were diluted in TBS-T containing 5% low-fat milk powder. Membranes were washed in TBS-T for 21 min (3 X 7 min) before and after incubation with secondary antibodies. Proteins were detected using enhanced chemiluminescence (ECL) (BioRad) and visualised with the ChemiDoc (BioRad; USA) and analysed via densitometry (ImageLab 5.2.1, BioRad; USA). Cell lysates were normalised to automated total protein measurement via ChemiDoc stain-free detection software.

Whole cell lysates were prepared by dissolving cells in Laemmli Sample Buffer containing 5% of 2-mercaptoethanol (Bio-Rad). Antibodies used for immunoblotting include: anti-progerin^{26 (link)} with 1:500 dilution, and mouse anti-GAPDH (sc-47724; Santa Cruz) with 1:3000 dilution. Protein samples were loaded on 12% polyacrylamide gels (Bio-Rad) and run at 100 V until the dye front reached the bottom of the gel. The proteins were transferred onto 0.45 μm pore-size nitrocellulose membranes (Bio-Rad) using Turboblot (Bio-Rad). Then, blots were probed with primary and secondary antibodies, and images were developed using ECL (Bio-Rad). Image Lab software (Bio-Rad) was used for image capture and analysis. For each experiment, all conditions (control, DMSO vehicle and Everolimus does) run on the same gel. For each gel, values are normalized to control and then the results of the different gels averaged. Complete gels are shown in Fig. S2.

Tumor cells were homogenized in lysis buffer: PBS containing 10% Triton X-100 (Sigma) with cOmplete™ Protease Inhibitor Cocktail (Roche) for 30 min in ice. Samples were then centrifuged for 15 min at 10,000 rpm 4°C. Protein concentration in the resulting supernatants was quantified using Protein Assay Dye Reagent Concentrate (BioRad) diluted in deionized water. Equal amounts of lysates were fractionated by BioRad mini-PROTEAN TGX 4-15% gels (BioRad) for SMG1 WB or 10% SDS-PAGE for UPF1 and STAT3 and electrotransferred to 0.45μm pore size nitrocellulose membranes (BioRad). After blocking with TBS (BioRad)/0.1% Tween (Sigma)-20/5% milk, the membranes were probed with rabbit anti-mouse SMG1 (Cell Signaling; 1:1,000; clone Q25), rabbit anti-mouse UPF1 (Sigma; 1:200; Polyclonal), mouse anti-mouse STAT3 (Cell Signaling; 1:1,000; clone 124H6), and rabbit anti-mouse β-Actin (Cell Signaling; 1:2,000; clone 13E5) o/n in agitation at 4°C. HRP-linked anti-rabbit or anti-mouse antibody (both from Cell Signaling; 1:5,000) were used as secondary antibodies. Protein bands were detected by chemoluminiscence using Amersham™ ECL™ Western Blotting Detection Reagents or Amersham™ ECL™ Prime Western Blotting Detection Reagents (GE Healthcare) in a ChemiDoc device (BioRad).