Chemidoc mp imaging

Cells were washed with iced‐cold PBS and lysed with 2% SDS buffer (50 mM Tris–HCl, pH 6.8%, 2% SDS, and 10% glycerol) supplemented with protease and phosphatase inhibitors (Roche). The protein concentration was measured using the DC protein assay (Bio‐Rad). Equal amounts of whole cell lysates (10–20 μg) were loaded onto 4%–20% Mini‐PROTEAN TGX Precast Gels (Bio‐Rad). After blocking with polyvinylidene difluoride (PVDF) blocking reagent for Can Get Signal (Toyobo Life Science), the blots were incubated overnight with the indicated primary antibodies: anti‐CALML5 antibody (ab154631) used at 1:1000 dilution and anti‐actin antibody (A5316) used at 1:5000 dilution. The membranes were incubated with the appropriate horseradish peroxidase‐conjugated secondary antibody (diluted 1:3000) (GE Healthcare), and this was followed by detection with enhanced chemiluminescence (ECL; GE Healthcare). All dilutions were made in Can Get Signal Immunoreaction Enhancer Solution (Toyobo Life Science). Images of the western blot signals were acquired by Chemidoc and Chemidoc MP imaging systems with Image Lab Touch Software (Bio‐Rad).

Equivalent amounts of brain or vesicle protein (determined by BCA assay Pierce) were electrophoresed on 4–12% Bis-Tris gels (NuPage; ThermoFisher) or 4–20% Criterion© TGX stainfree precast gels (Biorad) and then transferred onto nitrocellulose membrane (Biorad). Membranes were probed with primary antibody diluted in PBS-T overnight at 4°C and then with HRP secondary antibody (Amersham) for 1 h. Immunoreactivity was detected using enhanced chemiluminescence (ECL) solutions (Biorad) which was visualized using a Biorad ChemiDoc™ MP imaging system. To allow for visualization of total protein and ensure equal loading in the absence of an exosomal house-keeping protein, gels (Biorad) were activated by UV light to allow trihalo compounds within the gel to react with tryptophan residues of proteins in a UV-induced reaction to produce fluorescence which was visualized using a Biorad ChemiDoc™ MP imaging or membranes were stained with ponceau S (1% (w/v) ponceau S in 5% acetic acid). Primary antibodies used in this study were syntenin (Abcam EPR8012 ab133267) and calnexin (Abcam ab22595).

Total protein was extracted from tissues/cells using RIPA lysis buffer (Beyotime, Shanghai, CN), containing protease and phosphatase inhibitors cocktails. The BCA Protein Assay Kit (Beyotime) was used to measure protein concentrations according to the manufacturer’s protocol. An identical protein amount (20  $\mu$ g) was separated using SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Subsequently, 5% skim milk was used to block the activated membrane for 1 h, and then the membrane was incubated at 4^∘C with primary antibodies against FILIP1L (ab122835, Abcam, UK), matrix metalloproteinase-9 (MMP9, GeneTex, GTX31891, USA), and matrix metalloproteinase-2 (MMP2, GeneTex, GTX55708, USA). $\beta$ -actin (Sigma Aldrich, A2228, USA) was used as the internal control. Subsequently, the blots were incubated with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then were visualized using enhanced chemiluminescence western blotting detection reagents (Millipore, Billerica, MA, USA). Finally, photographs were taken using ChemiDoc™MP Imaging (Bio-Rad).