Quickblocktm western buffer

The protein concentration was measured by a BCA Protein Assay Kit (Beyotime; Shanghai, China; Cat. No. P0010S). Total proteins were dissolved using 6–12% SDS-PAGE and transferred to the polyvinylidene difluoride membranes (Millipore, Massachusetts, USA). The membranes were then blocked with QuickBlockTM western buffer (Beyotime; Cat. No. P0231) for 20 min at room temperature and incubated with primary antibody including CD63 (Abcam), CD9 (Diagbio; Hangzhou, China; Cat. No. db919), ALIX (Diagbio; Hangzhou, China; Cat. No. db3856), HSP70 (Diagbio; Hangzhou, China; Cat. No. db2396), Calnexin (Abclonal, Wuhan, China; Cat. No. A0803), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Diagbio; Hangzhou, China; Cat. No. db106), and GAS1 (absin, Shanghai, China; Cat. No. abs141177) at 4 °C overnight. Following, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abclonal, Wuhan, China; 1:3000) at room temperature for 2 h. The blots were detected using BeyoECL Plus (Beyotime; Shanghai, China; Cat. No. P0018S).

The honeybee whole bodies were lysed in RIPA buffer (pH 7.5). The lysate was centrifuged at 10,000 × g for 15 min at 4°C. The supernatant was then collected, and the protein concentration was determined using the BCA Protein Assay Kit (Beyotime, China). The extracted protein was separated on a 10% SDS-PAGE gel and transferred onto PVDF membranes (Millipore, Bedford, MA, United States). The membrane was blocked with QuickBlock^TM Western buffer (Beyotime, China) for 1.5 h to reduce non-specific binding. Next, the blot was incubated with the primary antibody overnight at 4°C. After washing, the blot was incubated with AP-labeled goat anti-mouse IgG (H + L) secondary antibody (Beyotime, China) for 4 h at 4°C. Finally, the signal was detected using an enhanced BeyECL Plus kit (Beyotime, China) and visualized in Fusion Fx by Vilber Lourmat. The optical density of each band was quantified using Fusion Capt Advance Fx7 software (Beijing Oriental Science, China), using tubulin as an internal control. The antibodies used included anti-AccCYP314A1 (1:100), anti-AccCYP4AZ1 (1:100), anti-AccCYP6AS5 (1:100), and anti-tubulin (1:1,000).