Benchmark protein standard

Cells were washed once with PBS and lysed in LDS buffer (141 mM Tris, 2% (w/v) LDS, 10% (v/v) glycerol, 0.51 mM EDTA, 0.22 mM G250, 50 mM DTT, 50 mM TCEP, 50 mM chloroacetamide, pH 8.5) on ice for 15 min, tip sonicated, and heated for 10 min at 95 °C. Then, 5 μL of samples and BenchMark protein standard (Invitrogen, Waltham, MA, USA) were subjected to PAGE (NuPAGE 4–12% Bis-Tris gels, 140 V). The gels were stained with SYPRO ruby (ThermoFisher Scientific, Waltham, MA, USA) according to manufacturer’s instructions (quick protocol). The gels were imaged at a resolution of 200 μm, with 470 nm excitation and 610 nm emission filters set using a Typhoon Trio (GE Healthcare, Chicago, IL, USA). The protein concentration in each sample was determined by relative densitometry compared to the standard using ImageQuant analysis toolbox software (GE Healthcare, Chicago, IL, USA).

Vesicle pellets from individual fractions were lysed with 30 μl LDS buffer (141 mM Tris pH 8.5, 2% (w/v) LDS, 10% (v/v) glycerol, 0.51 mM EDTA, 0.22 mM G250, 50 mM DTT), and denatured at 95°C for 5 min. A 5 μl aliquot from every sample, and 5 μl BenchMark protein standard (Invitrogen) was separated by PAGE (NuPAGE 4–12% Bis-Tris gels), and the gels stained with SYPRO ruby (ThermoFisher Scientific) according to manufacturer’s instructions. Gels were imaged using a Typhoon Trio (GE Healthcare) at a resolution of 200 μm, and excitation (470 nm) and emission filters (610 nm) set. The protein concentration in each fraction was determined by relative densitometry of each lane compared to the BenchMark standard, as calculated by ImageQuant analysis toolbox software (GE Healthcare).