Protein Aggregation Assays

Protein aggregation was measured using Proteostat (Enzo #ENZ-51023) following manufacturer’s instructions. Ventricular myocardial lysate (Cell Signaling lysis buffer) was obtained, protein concentration assayed, and 10 μg protein loaded into a 96 well microplate and protein aggregates analyzed using the Proteostat® assay kit (Enzo Life Sciences) following manufacturer’s instructions. After background subtraction, values were normalized to WT sham.

For a second assay, 1 mmol/L phenylmethanesulfonyl fluoride and a phosSTOP™ tablet (Sigma-Aldrich/Roche) were added to myocardial lysates. This was centrifuged at 8,000g in 4°C for 10 minutes, supernatant (soluble fraction) extracted, and the pellet then resuspended in insoluble extraction buffer (40 mmol/L Tris HCl [pH 8.8], 1% SDS, 8% glycerol), boiled for 5 minutes, and re-centrifuged at 3,000g for 5 minutes. 2.5 μg of the supernatant (insoluble fraction) was filtered through nitrocellulose membrane (pore diameter, 0.22 μm, Millipore) using a dot-blot apparatus (BioRad), and immunoprobed for ubiquitin and α-tubulin.