Bca assay

30 µg of cell culture media were first measured by BCA assay (Thermo Fisher Scientific Inc., Waltham, USA) and then mixed with 7.5 µl of 4x Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, USA) in a final volume 30 µl. 30 µl of cell culture media proteins with final 1x Laemmli Sample Buffer were boiled at 70°C for 10 min and sonicated at 4°C for 5 min. The sonicated protein extracts were further sent to the proteomics core facility (Bavarian Center for Biomolecular Mass Spectrometry).
25 µl EVs were first mixed with 25 µl 2x RIPA buffer (Abcam plc, Cambridge, UK; ab156034) and stored at -80°C for overnight incubation. Next, samples were boiled at 70°C for 10 min and sonicated at 4°C for 5 min. The sonicated protein extracts were centrifuged at 10,000 rcf at 4°C for 30 min. Protein concentration was measured by performing a BCA assay (Thermo Fisher Scientific Inc., Waltham, USA). 2 µg of EV proteins were further mixed with ddH₂O and 5 µl of 4x Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, USA) with 2-mercaptoethanol (Merck KGaA, Darmstadt, Germany) in a final volume of 20 µl. 20 µl of EV proteins with 1x Laemmli Sample Buffer were sent to the proteomics core facility (Bavarian Center for Biomolecular Mass Spectrometry).

The cell pellets were dissolved and further digested following a previous protocol with slight modifications [33 (link),34 (link)]. Typically, the cell pellet was first dissolved in denaturing solution (10 mM EDTA and 0.2% SDS in 40 mM Tris at pH 8.0) and boiled for 10 min. The protein amount was quantified by a BCA assay (Life Technologies, Thermo Fisher Scientific, Waltham, MA, USA). Then, TCEP was introduced at a final concentration of 10 mM, followed by adding powdered urea to the cooled sample to 8 M, and the solution was incubated at 37 °C for 30 min with end-to-end rotation. Disulfide bond formation was prevented by alkylating the cysteine residues with 15 mM iodoacetamide (IAA) in the dark at room temperature with an end-to-end rotation for 30 min. The reaction was halted with the addition of 20 mM DTT at room temperature for 15 min. The alkylated sample was diluted 10-fold with PBS prior to trypsin (Pierce, Cat. 90057, Thermo Fisher Scientific, Waltham, MA, USA) addition at a 1:20 enzyme-to-protein ratio. The digestion took place at 37 °C with end-to-end rotation overnight or for defined hours. The efficiency of digestion was evaluated by silver staining with 15% SDS-PAGE. The digested samples were further cleaned by an MCX column (Waters, MA, USA) and dried by a SpeedVac (Thermo Fisher Scientific, Waltham, MA, USA).

CD1 mouse embryos were surgically collected from the uterus at the indicated time points, and hearts were dissected under the microscope. The dissected hearts were washed twice with ice-cold PBS and lysed in cell lysis buffer containing 1 × protease inhibitor (Roche), followed by homogenization. Cultured cells were washed twice with ice-cold PBS, and were directly lysed in cell lysis buffer. Human heart tissues were flash frozen in liquid nitrogen, and then homogenized into small pieces in liquid nitrogen. Tissue pieces were lysed in lysis buffer and sonicated. All lysates were subsequently sonicated and quantified by BCA assay (Life Technologies). Forty micrograms of protein was loaded and separated by SDS-PAGE on 4–12% Bis-Tris gels (Life Technologies) and transferred to nitrocellulose membranes. Membranes were blocked with 5% milk in TBS-T and then incubated with primary antibodies overnight at 4 °C. The next day, membranes were incubated with the appropriate secondary antibodies and developed using ECL Detection Kit (GE Healthcare Bio-Sciences).

Western blot analysis was performed on wild type and CRND8 mice of 1, 4 and 9 months of age (3–4 animals per condition per age, see figure legend) to investigate changes in synaptic, inflammatory, and glial cell protein expression. Cortices from wild type and CRND8 mice were dissected and homogenized in RIPA buffer (1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 10% glycerol, 20 mM Tris pH 8.0, 150 mM NaCl and 1 mM EDTA supplemented with 1 μg/ml each of leupeptin, aprotinin, pepstatin, 10 mM NaF, 1 mM sodium ortho-vanadate and 1 mM PMSF) using a Dounce homogenizer and lysed on ice for 30 minutes. Lysates were centrifuged at 13,000 rpm for 10 min at 4 °C to pellet cell debris and protein concentration was determined using a BCA assay (Life Technologies, Burlington, Ontario, Canada). Supernatants were diluted with 3X sample buffer, resolved by SDS-PAGE, and analyzed by immunoblotting using antibodies as detailed in the figure legend and Table 2. Differences between samples were assessed using a 1-sample t test.