Femto extended duration substrate

SR‐related proteins were analyzed by Western blotting as previously described (Eshima, Tamura, et al., 2017 (link)). The protein abundance of the ryanodine receptor (RyR), the dihydropyridine (DHPR), the calsequestrin (CSQ), and the SR Ca²⁺‐ATPase (SERCA) was assessed. Briefly, polyvinylidene fluoride membranes were incubated overnight at 4°C with the following primary antibodies: anti‐type 1 ryanodine receptor (RyR) antibody 34C (MA3‐925; Thermo Fisher Scientific); anti‐dihydropyridine (DHPR) antibody 20A (ab2864; Abcam); anti‐calsequestrin antibody VIIID12 (MA3‐913; Thermo Scientific); anti‐SR Ca²⁺‐ATPase 2 (SERCA2) antibody 2A7‐A1 (MA3‐919; Thermo Scientific), and anti‐ glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) antibody 14C10 (no. 2118; Cell Signaling Technology) at 4°C. The membranes were incubated with the appropriate secondary antibody conjugated to horseradish peroxidase, enhanced by SuperSignal West Dura and Femto extended duration substrate (Thermo Fisher Scientific), and quantified by densitometry (C‐DiGit, LI‐COR Biosciences).

Extracellular vesicles isolated by differential centrifugation were suspended in 250 μL PBS-2.5 M sucrose, loaded in a SW60 tube and overlaid with 15 successive 250 mL layers of 20 mM Tris pH 7.4 containing decreasing concentrations of sucrose (from 2 to 0.4 M). Tubes were centrifuged for 16 h at 200,000×g at 4 °C. Fractions of 250 μL were collected and sucrose density was measured using a refractometer. Fractions were mixed 1:1 with Laemmli sample buffer and incubated for 5 min at 95 °C, followed by SDS-page and Western blotting analysis using standard procedures. In brief, proteins were transferred to polyvinylidene difluoride (PVDF) membrane (Millipore) and incubated with the following antibodies: mouse anti-CD9 (1:1000; Biolegend), mouse anti-CD63 (1:1000; Abcam). Membranes were washed, incubated with appropriate peroxidase-conjugated secondary antibodies and developed by SuperSignal West Dura or Femto Extended Duration Substrate (ThermoFisher).