Rabbit anti mfn1

Specific proteins expressed in the mouse brain and neuron cell cultures were determined by western blotting using specific antibodies. Proteins (40 µg) were separated on 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and then transferred onto a nitrocellulose membrane. Nonspecific binding sites were blocked using 5% fat-free dried milk in 1 × TBST at 37 °C for 1 h. The membrane was incubated at 4 °C overnight with individual primary antibodies, including rabbit anti-OPA1 (Cell Signaling Technology, USA), rabbit anti-MFN1 (Proteintech, USA), mouse anti-MFN2 (Abcam, USA), rabbit anti-cytochrome c (Cell Signaling Technology, USA), rabbit anti-cleaved caspase 3 (Cell Signaling Technology, USA), mouse anti-GAPDH (Proteintech, USA), rabbit anti-β-actin (Proteintech, USA), and rabbit anti-VDAC (Cell Signaling Technology, USA). After washing with TBST, the membrane was incubated with secondary antibodies, goat anti-mouse IgG or anti-rabbit IgG conjugated to horseradish peroxidase (1:10,000). The blots were visualized using a Chemiluminescent Imaging System (Tanon Science and Technology).

Rat insulinoma cell line (INS-1) was from the American Type Culture Collection (Manassas, VA, USA). Rabbit anti-Bmal1 antibody was purchased from Abcam (Cambridge, MA, USA). Rabbit anti-Mfn1, anti-Mfn2, anti-Fis1, and anti-GAPDH antibodies were from Proteintech (Chicago, IL, USA). RPMI-1640 medium and cell culture reagents were from Gibco (Grand Island, NY, USA).