To acquire specimens for immunoblot, the mice were perfused with cold PBS and one side of the hippocampus was dissected immediately (Wang et al., 2021b (link)). Hippocampal tissue was homogenized at 4°C in RIPA buffer (containing protease and phosphatase inhibitors) and followed by sonication (10 s × three times with a 10-s interval). Ten micrograms of protein was loaded for immunoblot as previously described (Wang et al., 2021b (link)). GAPDH was used as a loading control for total lysate immunoblots. Primary antibodies used were as follows: p-DRP(Ser616) (Thermo Fisher; #PA5-64821, 1:500), DRP1 (Cell Signaling, #8570, AB_10950498, 1:1000), mitofusin-1 (Abcam, #104274, 1:500), mitofusin-2 (Cell Signaling, #9482, AB_2716838, 1:1000), p-MFF (Ser146) (Cell Signaling, #49281, 1:1000), MFF (Cell Signaling, #84580, AB_2728769, 1:1000), Tom20 (Cell Signaling, #42406, AB_2687663, 1:1000), VDAC1 (Santa Cruz, #sc-390996, 1:50), OXPHOS (Abcam, #ab110413, AB_2629281, 1:2000), and GAPDH (Cell Signaling #2118s; AB_561053, 1:1,000) overnight at 4°C followed by incubation with an IR-dye-labeled secondary antibody for 1 hour and measured with LiCor Odyssey followed by densitometric analysis.
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