Ab56889

For Western blot analysis, whole-cell homogenates were used. Harvested cells were resuspended in cold RIPA buffer (Thermo Fisher Scientific) supplemented with protease inhibitors cOmplete™ Protease Inhibitor Cocktail (Roche) and phosphatase inhibitors PhosSTOP EASYpack phosphatase inhibitors (Roche). Protein concentrations were assessed using a BCA Protein Assay Kit (Thermo Fisher Scientific), and 10 µg of total protein lysates were loaded per well on a NuPAGE 4–12% Bis-Tris SDS-PAGE gel (Thermo Fisher Scientific). After electrophoresis, proteins were transferred onto a PVDF membrane (BioRad) and probed with antibodies raised against MFN2 (Abcam, ab56889), TOM70 (Abcam, ab135602), SOD2 (Cell Signaling, 13141 S), Parkin (Abcam, 77924), and GAPDH (Millipore, MAB374). Subsequently, the blots were incubated with the corresponding secondary antibodies, and target proteins were detected by enhanced chemiluminescence using Clarity™ ECL Western Kit (BioRad). The chemiluminescence signal was detected using the ChemiDoc™ Touch Imaging System (BioRad) and quantified by densitometry using Image Lab 6.0 analyzer software (BioRad). Optical density values assessed for target proteins were normalized by the indicated loading control. For each figure, all blots were processed in parallel and originated from the same experiment.

Protein expression of mitochondrial fission marker, dynamin-related protein-1 (DRP-1); and fusion markers, mitofusin-1/2 (Mfn-1/2) was determined using Western blot analysis as described before (Solanki et al., 2015 (link)). Antibody dilutions: DRP-1 (ab156951, 1:1000), Mfn-1 (ab57602, 1:500) and Mfn-2 (ab56889, 1:500) were purchased from Abcam (Cambridge, MA). Anti-Secondary antibody (1:2000, Cell Signaling Technology, Danvers, MA) was used. β-actin was used as a loading control (sc-47778, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA).

Ventral midbrain was dissected from control and tamoxifen-injected iMfn2^DA mice and snap frozen in liquid nitrogen. Tissue was homogenized in RIPA buffer supplemented with protease inhibitors (Complete, Roche). Twenty micrograms of protein extracts were resuspended in Laemmli buffer, run on 12% SDS–polyacrylamide gel electrophoresis (Invitrogen) and then transferred onto polyvinylidene difluoride membranes (GE Healthcare). Blots were incubated overnight at 4°C with primary antibody against MFN2 (ab 56889, Abcam) and GAPDH (ab8245, Abcam). Immunodetection was performed according to standard techniques using enhanced chemiluminescence Immun-Star HRP Luminol/Enhancer (Bio-Rad).

Protein was extracted using RIPA (Beyotime, China) buffer supplemented with PMSF (Beyotime, China). Protein concentration was determined by BCA assay (Beyotime, China), and added into 5 × loading buffer and followed by boiling for 10 min. Equal amounts of proteins were separated on the 12% SDS-PAGE gel, electrotransferred onto PVDF membrane (Millipore, Billerica, MA, USA), and blocked with 5% skimmed milk or 5% BSA for 1 h at room temperature. Then the membrane was incubated with the corresponding primary antibody at 4 °C overnight and incubated with the secondary antibody for 1 h at room temperature. Detection was performed with the enhanced chemiluminescence (ECL) detection reagent. Relative protein level was standardized to GAPDH. The antibodies included DRP1 (1:1000, ab56788, Abcam, USA), OPA1 (1:1000, 612606, BD bioscience, USA), MFN1 (1:1000, ab57602, Abcam, USA), MFN2 (1:1000, ab56889, Abcam, USA), LCLAT1 (1:1000, ab153987, Abcam, USA), and GAPDH (1:5000, RK-200-301-A33, Lianke Biotech, China).

Expression of Mfn2 or Mlh1 in the mitochondria was performed in HRECs by immunofluorescence technique using antibodies against Mfn2 (Cat. No. ab56889, Abcam; 1:250 dilution) and Mlh1 (cat. no. ab92312, Abcam, Cambridge, MA, USA; 1:100 dilution), as previously described^{13 (link),14 (link)}. CoxIV was used as a mitochondrial marker; Cat. No. ab153709 for Mfn2 and Cat. No. ab33985 for Mlh1; both from Abcam, and at 1:250 dilution each. Secondary antibodies included Alexa Fluor-488 (green) conjugated anti-rabbit (Cat. No. Molecular Probes-Life Technologies, Grand Island, NE), DyLight 488-conjugated anti-mouse (Cat. No. DI-2488, Vector Laboratories, Burlingame, CA) and Texas red-conjugated anti-mouse (Cat. No. TI-2000, Vector Laboratories, Burlingame, CA); each at 1:500 dilution. Immuno-labelled cells were mounted using DAPI-containing (blue) Vectashield mounting medium (Vector Laboratories), and were examined under ZEISS (Carles Zeiss, Inc., Chicago, IL, USA) at 40X objective magnification with the Apotome module^{13 (link),14 (link)}. The images were calibrated with the ZEISS proinbuilt software package and modules, and the Pearson’s correlation coefficient was calculated using the colocalization software module^{13 (link)}.