Anti histone h3.3b

Total proteins were extracted from head tissue of Drosophila flies following treatment (100 flies per group). The removed tissue was homogenized in a buffer solution on ice for 1 h and was centrifuged at 4 °C for 13,000 rpm for 20 min. The supernatant was quantified using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). The proteins were separated on 12.5% or 15% sodium dodecyl sulfate polyacrylamide gels (Bionovas Pharmaceuticals, Washington, DC, USA) and were transferred to polyvinylidene difluoride membranes (GE Healthcare Life Sciences, Barrington, IL, USA). The primary antibodies were anti-Histone H3.3B (Thermo Fisher Scientific) anti-pTau 181 and anti-Tau (Cell Signaling Technology, Danvers, MA, USA) antibodies. The secondary antibodies were horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), and protein immunoreactive bands were visualized using the enhanced chemiluminescence (ECL) substrate (Millipore, Billerica, MA, USA). Band intensities were quantified using Image J analysis software (version 1.48t, Wayne Rasband, NIH, Washington, DC, USA).

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).