Anti p drp1

Cultured neurons in 6-well plates were harvested at 10 weeks of differentiation as cell pellets and were resuspended in RIPA buffer (BIOSESANG) containing protease inhibitors (ThermoFisher) and phosphatase inhibitors (ThermoFisher). They were then chilled on ice for 30 min and sonicated. The supernatants were collected after centrifugation at 13,000 rpm for 30 min to remove membrane lipids. Protein concentration was determined using a BCA Protein Assay Kit (ThermoFisher), and 25 ug of protein for each sample was electrophoresed on 8% or 12% gels. The separated samples were transferred onto a PVDF membrane and incubated with target antibodies. Protein bands were visualized by Immobilon Western (Millipore) and were detected using Chemi-Doc (Bio-Rad). The following primary antibodies were used: Tau5 anti-tau (1:1,000, ThermoFisher), AT8 anti-p-tau (1:1,000, ThermoFisher), anti-OPA1 (1:1,000, Santa Cruz), anti-Mfn1 (1:1,000, Abcam), anti-Mfn2 (1:1,000, Cell Signaling), anti-Drp1 (1:1,000, Cell Signaling), anti-Fis1 (1:1,000, Santa Cruz), anti-Ub (1:4,000, Santa Cruz), anti-LC3B (1:1,000, Cell Signaling), anti-LAMP2 (1:1,000, Santa Cruz), anti-Beclin1 (1:1,000, Cell Signaling), p62 anti-SQSTM1 (1:1,000, Santa Cruz), 4G8 anti-APP (1:500, Covance), anti-p-Drp1 (Ser637, 1:1000, Cell Signaling) and anti-β-actin (1:10000, Santa Cruz).

The western blotting procedures were fully described previously.
¹⁴ (link) The following primary antibodies were used: anti‐4‐HNE (Abcam, AB46545, 1:4000), anti‐Bax (Santa Cruz, Sc‐7480, 1:100), anti‐Bcl‐2 (Santa Cruz, Sc‐7382, 1:200), anti‐catalase (GeneTex, GTX110704, 1:1000), anti‐citrate synthase (Santa Cruz, Sc‐390693, 1:200), anti‐p‐DRP1 (Ser616, Cell Signaling, #34555, 1:500), anti‐DRP1 (Cell Signaling, #5391S, 1:500), anti‐Fis1 (Santa Cruz, Sc‐376447, 1:200), anti GPx1 (Thermo Fisher Scientific, PA5‐26323, 1:1000), anti‐MFN2 (Santa Cruz, Sc‐100560, 1:200), anti‐Mul1 (Abcam, AB209263, 1:1000), anti‐OPA1 (Thermo Fisher Scientific, MA5‐16149, 1:1000), anti‐Parkin (Abcam, AB77924, 1:2000), anti‐PINK1 (Thermo Fisher Scientific, PA1‐16604, 1:500), anti‐PGC‐1α1 (Millipore, AB3242, 1:1000), anti‐SOD2 (Genetex, GTX116093, 1:1000) and anti‐VDAC (Santa Cruz, Sc‐390996, 1:200). Then, anti‐rabbit (Cell Signaling, #7074S, 1:4000) or anti‐mouse (Cell Signaling, #7076S, 1:4000) secondary antibodies were used. The blots were revealed using a Pierce ECL kit (Thermo Fisher Scientific) or SupraSignal Femto kit (Thermo Fisher Scientific), and proteins were visualized by enhanced chemiluminescence (iBright 1500 Imaging System, Invitrogen) and quantified with ImageJ Software (version 1.8.0). Ponceau coloration was used as the loading control.