Ab90787

Immunoprecipitations were performed using Dynabeads (Thermo Fisher Scientific, York, UK). Antibodies (10 μg) were coupled to M270 beads according to the manufacturer's protocol. Beads were washed in C1 buffer then C1-diluted antibodies added. Buffer C2 was added and reactions incubated for 18 h at 37 °C. HB and SB washes contained 0.1% Tween-20 (Sigma-Aldrich, Dorset, UK). Before use, beads were washed in PBS containing 0.1% BSA. Lysates were prepared in kit extraction buffer supplemented with EDTA-free protease inhibitor cocktail tablets (Roche Diagnostics Ltd., West Sussex, UK) and 100 mM NaCl. Immunoprecipitations were performed for 30 minutes at 4 °C, then beads were washed and eluted. Antibodies were ab90787, PGK1; ab18257, DJ1; and ab13579, TRF2 (Abcam, Cambridge, UK). Experiments were performed twice.

Cells were pre-cultured overnight, diluted to OD₆₀₀ = 0.1, and grown until mid-exponential phase (OD₆₀₀ ~ 0.5) in YPD at 22 °C (Hsp70 mutant strains and control) or 30 °C. Cells were harvested as 2 OD₆₀₀ units, resuspended in 200 µl of 0.2 M NaOH, and incubated for 20 min on ice. Cell pellets were resuspended in 50 µl Laemmli sample buffer (Bio-Rad) supplemented with β-mercaptoethanol and boiled for 5 min at 95 °C. 25 µl supernatant was typically loaded per lane on a 4–15% precast polyacrylamide 18 well Criterion TGX protein gel (Bio-Rad). Gel transfer onto PVDF membranes was performed using a wet blotting system (Criterion, Bio-Rad). Membranes were probed with anti-GFP (ab6556, Abcam, 1/10,000 dilution) and anti-Pgk1 (ab90787, Abcam, 1/10,000 dilution) as a loading control overnight at 4 °C. As secondary antibodies, goat anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680RD were used (LI-COR; 1/20,000 dilution) and membranes were scanned using the LI-COR Odyssey Infrared scanner. Hsp104-GFP-Pea2 protein levels were calculated by measuring the background corrected band intensities using Fiji/ImageJ 2.1.0/1.53c software and normalizing to Pgk1 protein levels. The adjusted Hsp104-GFP-Pea2 protein level of the respective control strain was set to 1. Four independent experiments were performed. Uncropped Western blots are shown in Supplementary Fig. 8.

The aortic tissue homogenates were mixed with a 2 × solution of sample buffer (62.5 mM Tris–HCl, pH 6.8, containing 2% (w/v) SDS, 25% (w/v) glycerol, 5% (v/v) β-mercaptoethanol, and 0.01% (w/v) bromophenol blue) and heated at 95 °C for 3 min. Next, 40 μg of protein sample was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore-Linco, St Charles, MO). Membranes were blotted for 1 h in 5% (w/v) fat free milk and incubated overnight at 4 °C with primary antibodies against eNOS (1:1500, ab5589), p-eNOS (1:1000, ab184154), PKG1 (1:2000, ab90787, Abcam, Cambridge, UK), and β-actin (1:3000, Bioworld Technology, Nanjing, China). The bound antibody was detected using a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. Bands were visualized by enhanced chemiluminescence (ECL, Millipore-Linco, St. Charles, MO) and analyzed using ImageJ (National Institutes of Health, Bethesda, Maryland, USA). All protein expression was normalized to that of β-actin, which served as an internal control.