Enhanced chemiluminescence ecl substrate

Total proteins were extracted from the head tissue of Drosophila following the treatment described (100 files for each group). The removed tissue was homogenized in a buffer solution that was placed on ice for one hour and then centrifuged at 4 °C for 13,000 rpm for another 20 min. The separated solution was quantified by using a BCA protein assay kit (Thermo Fisher Scientific Inc. Waltham, MA, USA). Proteins were separated on 12.5% or 15% SDS polyacrylamide gels (Bionovas Pharmaceuticals Inc., Washington, DC, USA), and proteins were transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Barrington, IL, USA). The antibodies used in this study were anti-Histone H3.3B (Thermo Fisher Scientific Inc.), and anti-amyloid-beta (anti-Aβ) (Covance Cat#SIG-39220, BioLegend, Dedham, MA, USA). Antibodies were detected by suitable horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology Inc.), and then proteins’ immunoreactive bands were visualized by the enhanced chemiluminescence (ECL) substrate (Millipore, Billerica, MA, USA), and the band intensities were quantified with the Image J analysis software (version 1.48t, Wayne Rasnabd, USA).

Total proteins were extracted from mouse tissues or cells using RIPA Lysis Buffer (#P0013B; Beyotime), following the manufacturer's instructions. A total of 30 μg of protein from each sample were boiled at 100°C for 5 minutes, separated through SDS‐PAGE, and transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% lipid‐free milk for 1‐2 hours, incubated with primary antibodies diluted in TBST for 2 hours at room temperature, washed with TBST and incubated with secondary antibodies for 1 hour at room temperature. Protein abundances were determined by development with enhanced chemiluminescence (ECL) substrates (Millipore). GAPDH was used as an internal standard. Primary antibodies targeting c‐Kit (#ab256345; Abcam), P65 (#ab16502; Abcam), p‐P65 (#ab86299; Abcam), AKT (#9272; CST), p‐AKT (#4060; CST) and GAPDH (#ab8245; Abcam) were applied.

The cells with different treatments were lysed in RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) containing a protease inhibitor cocktail (Complete, Roche, Germany). Twenty micrograms of total protein from exosome lysate were loaded and separated on a standard sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA). The membranes were incubated with primary antibodies, followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device (CCD) camera-based imager (GE Healthcare Life Sciences, USA) after membrane incubation with enhanced chemiluminescence (ECL) substrates (Millipore, USA). The levels of specific protein were normalized to β-actin. The ImageJ software was used for image acquisition and densitometric analysis of the immunoblots. All results were obtained in three independent experiments and the data are presented as the mean ± SD. An unpaired, two-tailed Student’s t-test was performed for between-group comparisons using GraphPad Prism software version 8. All results of densitometric analysis are presented in Additional file 2.

The cells with different treatments were lysed in RIPA buffer (25 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) containing a protease inhibitor cocktail (Roche, Germany). Then, 20 μg of total protein from exosome lysate was loaded onto and separated on a SDS-PAGE gel and transferred to a polyvinylidene difluoride membrane (Millipore, USA). The membranes were incubated with primary antibodies, followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device camera-based imager (GE Healthcare Life Sciences, USA) after membrane incubation with enhanced chemiluminescence (ECL) substrates (Millipore, USA).

The cells with different treatments were lysed in RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS) containing a protease inhibitor cocktail (Complete, Roche). Twenty micrograms of total protein from exosome lysate were loaded and separated on a standard sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore). The membranes were incubated with primary antibodies, followed by peroxidase-conjugated secondary antibodies. The results were detected using a charge-coupled device (CCD) camera-based imager (GE Healthcare Life Sciences) after membrane incubation with enhanced chemiluminescence (ECL) substrates (Millipore). The levels of specific protein were normalized to β-actin. ImageJ software was used for image acquisition and densitometric analysis of the immunoblots. All results were obtained in 3 independent experiments, and the data is presented as the mean ± SD. An unpaired, two-tailed Student's t test was performed for between-group comparisons using GraphPad Prism software version 8. All results of densitometric analysis were presented in in Supplementary File 1.