Drp1 GTPase activity assay

Equal amount of protein samples (200 pg) of hippocampal CA1 from each group were immunoprecipitated with 5 μg of anti-Drp1 antibody and 30 μL of protein A/G-agarose (Santa Cruz). The beads were washed four times with GTPase wash buffer (50 mM Tris-HCl, pH 7.5, 1.0 M NaCl, 10 mM MgCl2, 4 mM DTT, 1 mM PMSF) and incubated with 0.5 mM GTP in reaction buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 20 mM EDTA, and 5 mM MgCl2) at 30° C for 1 h. The released free phosphate was measured using a PiColorLock Gold Colorimetric Assay Kit (#303-0030, Novus Biologicals) according to the kit booklet protocol. Absorbance was recorded at 660 nm on a microplate spectrophotometer (Bio-Rad, Benchmark Plus). A standard curve was prepared using the 0.1 mM Pi stock provided with the kit for quantification. Each sample was measured in triplicate using the kit, and values were calculated and presented as percentage changes compared to the sham GCI group.