Pierce lysis buffer

HEK cells were co-transfected with RFP-Rab14 and GFP-Cdc42 or GFP-Cdc42Q61L. 24 h after transfection, cells were lysed in Pierce Lysis Buffer (Thermo Scientific, Rockford, IL). Rabbit anti-Rab14 antibody was bound to Dynabeads (Life Technologies) and rabbit IgG was used as the control. After binding, the bead-antibody complex was mixed with cell lysates and incubated on a rotator at 4 °C for 1 hour. Beads were washed and eluted with SDS-PAGE sample buffer and immunoblotted with anti-GFP antibody.

Cells were grown to 70% confluence in T-75 culture flasks (Thermo Fisher Scientific). Prior to lysis, growth medium was removed, and cell monolayer was washed with 10 mL PBS (Corning). Cells were trypsinized (Corning) with 1 mL trypsin/flask and collected in 15 mL conical tubes. The cells were then pelleted by centrifuging for 5 minutes at 300×g (NuAire Awel C48). The supernatant was aspirated, and the cells were washed with 10 mL ice-cold PBS. The pellet was spun down again for 5 minutes at 300×g after which the PBS wash was aspirated. A 1 mL aliquot of lysis buffer was prepared by mixing cold Pierce lysis buffer (Thermo Fisher Scientific, 87787) was supplemented with 10 μL 100× protease inhibitor cocktail (Thermo Fisher Scientific, 78442). The pellet was resuspended in 200 μL ice-cold lysis buffer and incubated on ice for 30 minutes. The lysed cells were centrifuged again for 5 minutes at 13000 g on a tabletop centrifuge (Eppendorf 5415D) and the supernatant was collected. The protein concentration of the lysate was estimated by a BCA assay (Thermo Fisher Scientific, 23225). Aliquots of cell lysate were stored at -20°C.

Total RNA was extracted and purified from EV pellets using the Trizol reagent according to the manufacturer’s protocol (Thermo Fisher). Protein was isolated from retinal tissue using Pierce lysis buffer (Thermo Fisher) with 1% protease inhibitor and 1% phosphatase inhibitor. For EV protein isolation, centrifuged and pelleted EVs were suspended in 200ul PBS. Protein concentrations were obtained using the bicinchoninic acid assay (BCA) assay (Thermo Fisher). Primary antibodies were mouse anti-γ-catenin (Santa Cruz, sc-33634, 1:1000), rabbit anti-actin (Cell Signaling, #8456, 1:5,000), mouse anti-CD81 (Santa Cruz, sc-166028, 1:1000), rabbit anti-CD63 (Novus, nbp2-67425, 1:1000). Blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, Millipore). Band density was quantified using Fiji/ImageJ (NIH).

Immunoprecipitation assays were performed by transfecting a bait GFP fusion protein into HeLa cells as discussed above. Approximately eight million cells were harvested for each sample and lysed into Pierce lysis buffer (Thermo) containing Complete protease inhibitors (Roche). Lysate was clarified by centrifugation at 10,000 × g for 30 min. Pellet and a portion of supernatant were reserved as input. The remaining lysate was incubated with 4 µg of goat anti-GFP overnight, followed by immunoprecipitation with Protein G Magnetic SureBeads (BioRad). Beads were washed three times in Pierce lysis buffer and eluted into NuPAGE Sample Buffer (Invitrogen). The resulting eluent was run onto a NuPAGE 4–12%, Bis-Tris gel (Thermo), transferred to nitrocellulose and probed with primary antibody to be visualized with horseradish peroxidase conjugated secondary antibody. The antibodies used were NOLC1 antibody (NovusBio NBP1-2298), GFP antibody (Invitrogen A-11122), and anti-rabbit IgG Horseradish Peroxidase-Linked Species-Specific Whole Antibody (GE HealthCare NA934).

2 × 10⁷ RBE and SSP-25 PBS-washed cells were harvested and lysed with Pierce lysis buffer (Thermo, cat#8990) containing protease inhibitors cocktail (TargetMol, cat#C0001). Then incubated with NOTCH1 (CST,cat#3608), MFAP5 (abcam,ab203828) antibody and control IgG (CST,cat#3900) after centrifugation respectively. Subsequently, the cell lysates were incubated with Protein A/G Dynabeads (Thermo, cat#10002D/10004D). Afterwards, the Protein A/G Dynabeads were eluted and collected. The eluent was boiled and denatured for Western-Bolt.