Western blot analysis
Cells or fly brain tissues were lysed in RIPA buffer (Beyotime
Biotechnology) for 1 h on ice and centrifuged at 14,000 rpm to obtain
the supernatants, which were measured to detect protein concentration
quantified with a BCA protein assay kit (Beyotime Biotechnology). Total
protein (30μg) was resolved on 10% or 12% sodium dodecyl sulfate
polyacrylamide gel electrophoresis to separate different sizes of
proteins, followed by eletrotransfer onto nitrocellulose membranes. The
membranes were blocked in 5% non-fat milk and probed with primary
antibodies overnight at 4°C. Proteins were detected using a
chemiluminescent imaging system (FluorChem, ProteinSimple, San Jose, CA,
USA), after incubating with the appropriate secondary antibodies for 1 h
at room temperature (Huang et al., 2018).