P mff

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.

Proteins from lysed cells were fractionated via sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Nonspecific binding sites were blocked with 5% bovine serum albumin in Tris-buffered saline with Tween 20 (TBST: 120 mM Tris-HCl [pH 7.4], 150 mM NaCl, 0.05% Tween 20) for two hours at room temperature. The blots were incubated with specific primary antibodies (each diluted 1:1000) overnight at 4 °C. β-actin was used as a loading control. The membranes were then washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized using the Immobilon^™ Western Chemiluminescent HRP substrate (Millipore, USA). The following antibodies were obtained from Abcam: ET-1 (#ab178454), eNOS (#ab199956), p-eNOS (#ab215717), Fak (#ab40794), Src (#ab133283), Drp1 (#ab184247), Fis1 (#ab156865), GAPDH (#ab8245), Tom20 (#ab186735), DNA-PKcs (#ab32566), F-actin (#ab130935) and Met (#ab51067). The following antibodies were obtained from Cell Signaling Technology: p-Drp1 (#4494), Mff (#86,668) and p-Mff (#49,281).