P drp1 ser616

Immunofluorescence staining was carried out in animals as reported previously [19 (link)]. Briefly, free-floating sections (thickness = 30 μm) of the hippocampus were incubated with a rabbit polyclonal antiserum against p-Drp1 (Ser616) (Cell Signaling) and a mouse monoclonal antiserum, neuron-specific nuclear protein (NeuN, Chemicon). Two secondary antibodies were used that included a goat anti-rabbit IgG-conjugated with Alexa Fluor 488 for p-Drp1(Ser616) and a goat anti-mouse IgG conjugated with Alexa Fluor 568 for NeuN (Molecular Probes, Eugene, OR, USA). The merged images indicated the presence of p-Drp1(Ser616) immunoreactivity in the cytosol and NeuN in the nucleus of neurons. For double immunofluorescence staining of p-Drp1(Ser616) and COXIV, the sections of the hippocampus were first incubated with a rabbit polyclonal antiserum against p-Drp1(Ser616) (Cell Signaling). The sections were subsequently incubated with a goat anti-rabbit IgG conjugated with Alexa Fluor 488 for p-Drp1(Ser616). After fixed with 4 % paraformaldehyde for 5 min, the same sections were incubated with a polyclonal rabbit antiserum against COXIV (Cell Signaling) and then with DyLight 405-conjugated AffiniPure goat anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA, USA) for labeling COX IV.

Cells were washed with PBS and lysed in 1x RIPA buffer with protease and phosphatase inhibitors, with the addition of 1 U/ml Benzonase to degrade genomic DNA. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 hr at RT in LiCOR Odyssey blocking buffer. Blots were (Licor) and incubated overnight at RT with the following antibodies: IRF3 (Cell Signaling, 1:1000); pIRF3 Ser396 (Cell Signaling, 1:1000); STAT1 (Cell Signaling, 1:1000); pSTAT1 Tyr701 (Cell Signaling, 1:1000); Beta Actin (Abcam, 1:2000), β-Tubulin (Abcam, 1:5000); DRP1 (Cell Signaling, 1:1000); pDRP1 Ser616 (Cell Signaling, 1:1000), TFAM (Millipore, 1:1000), VDAC (Protein Tech, 1:1000). Membranes were incubated with appropriate secondary antibodies for 2 hr at RT prior to imaging on a LiCOR Odyssey Fc Dual-Mode Imaging System.

IP/WB analysis was carried out as described previously.⁶ The antibodies against the following molecules were used for the analyses: Sirt6 (1:500; Abcam; #ab62739), Drp1 (1:1000; Abcam; #ab56788), mitofusin2 (Mfn2) (1:1000; Abcam; ab56889), mitochondrial fission protein 1 (Fis1) (1:1000; Genetex; GTX111010), p‐Drp1‐Ser616 (1:1000; Cell Signalling Technology; #3455), optic atrophy 1 (OPA1) (1:1000; ImmunoWay; YN2976), and p‐Drp1‐Ser637 (1:1000, Cell Signalling Technology; #4867).