Supersignal west pico chemiluminescent substrate reagent

Mosquito carcasses, 30 each, were homogenized by pellet pestle in 100 μl of lysis buffer [42 (link)]. Aliquots of mosquito protein samples were resolved using SDS-polyacrylamide gels (Bio-Rad) and transferred to PVDF membranes (Merck Millipore). Then, membranes were blocked with Starting Block T20 (PBS) Blocking Buffer (Thermo Fisher Scientific) and incubated with the primary antibody against HPX8C (1:3000). Following three washes with TBS containing Tween-20, the membranes were incubated with goat anti-rabbit horseradish peroxidase-conjugated antibody (Sigma, 1:10000). After washing four times with TBS containing Tween-20, membranes were incubated with SuperSignal West Pico Chemiluminescent Substrate reagent for 5 min (Thermo Fisher Scientific) before visualization. The antibody against α-tubulin (Cell Signaling Technology) was used as the loading control.

Exosomes isolated by Abs_MNWs were lysed in M-PER reagent (Thermo Fisher Scientific, Massachusetts, Waltham, USA). Protein samples (20 µg) were separated on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes (0.45 µm, Millipore). The membranes were blocked with 3% skim milk for 1 h at room temperature and probed with primary mouse anti-TSG101 (1:1000), rabbit anti-HSP70 (1:1000), rabbit anti-glyceraldehyde 3-phosphate dehydrogenase (1:1000), rabbit anti-CD9 (1:1000), rabbit anti-CD63 (1:1000), rabbit anti-CD81 (1:1000), and rabbit monoclonal anti-GAPDH (1:1000) for overnight. Following incubation, the membranes were incubated with an appropriate secondary antibody (goat anti-mouse IgG [1:3000] or goat anti-rabbit IgG [1:3000]) for 1 h. Blots were washed three times with TBST buffer after each incubation step and visualized using a SuperSignal^® West Pico Chemiluminescent Substrate reagent (34077, Thermo Scientific).

Proteins were extracted from the homogenized frozen brain tissue using TPER^® (Thermo Scientific) mixed with Halt^® protease inhibitor cocktail (Thermo Scientific). Protein concentrations were determined using a BCA Protein Assay kit (Thermo Scientific) and appropriate amounts of protein were mixed with sodium dodecyl sulphate (SDS) sample buffer [62.5 mM Tris-HCl pH 6.8, 25% (v/v) glycerol, 2% SDS (w/v), 0.01% (w/v) bromophenol blue, 5% (v/v) β-mercaptoethanol]. Lysates (20 μg per sample) were separated by 10% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE), and the resolved proteins were transferred onto nitrocellulose membranes (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Blots were blocked with 5% w/v non-fat milk in TBST (TBS containing 0.1% v/v Tween 20) for 1 h and incubated with a rabbit anti-LRP1 antibody (1:50,000, Abcam^®, Cambridge, MA, USA) overnight at 4 °C. After incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000, Thermo Scientific) for 1 h at 22 ± 2 °C, signals were detected using SuperSignal^® West Pico Chemiluminescent Substrate reagent (Thermo Scientific). Images were captured and analyzed with a LAS-4000 luminescent image analyzer (Fujifilm Life Science, Stamford, CT, USA)