Sc 33796

Immunoblotting was performed with anti- C-subunit of ATP synthase (SCMAs) (1/1000) (ab181243; Abcam), anti-VDAC (1/666) (PC548; Calbiochem), anti-heat-shock protein-60 (HSP60) (1/666) (ab46798; Abcam), anti-PINK1 (1/500) (sc-33796; Santa Cruz Biotechnology), anti-NDUFS1 (1/500) (sc-50132; Santa Cruz Biotechnology), anti-CDK5 (1/500) (sc-6247; Santa Cruz Biotechnology), anti-PFKFB3 (1/500) (H00005209-M08; Novus Biologicals), anti-p25/35 (1/666) (2680; Cell Signalling), anti-caspase-3 (1/2000) (9661S; Cell Signalling), anti-CLN7 (1/500) (donated by Dr. Stephan Storch), anti-Parkin (1/100) (sc-32282; Santa Cruz Biotechnology), anti-LC3B (1/1000) (2775; Cell Signaling), anti-GFAP (1/500) (G6171; Sigma), anti-β-Tubulin III (1/500) (ab18207; Abcam) and anti-β-Actin (1/30,000) (A5441; Sigma).

Cultured NRVCs were processed for Western blot analysis as described previously (Xu et al., 2012 (link), 2013 (link)). Briefly, NRVCs were collected in 1 × SDS, boiled for 10 min, loaded to polyacrylamide gel for electrophoresis, and then transferred to polyvinylidene difluoride membranes. The membranes were incubated with primary and secondary antibodies, and processed for chemiluminescent detection using Lumigen ECL Ultra (TMA-6 Lumigen, Southfield, MI, United States). The images were acquired by using Amersham Imager 600 and quantified with ImageJ. The antibodies against cleaved Caspase 3 (cCasp3) (#9664), β-Actin (#4967), microtubule-associated protein Light Chain 3 (LC3B, #3868), Parkin (#2132), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (#5147), Voltage Dependent Anion Channel 1 (VDAC1) (#4866), Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) (#4259) and Cytochrome c oxidase subunit 4 (COX IV) (#4850) were purchased from Cell Signaling Technology (Danvers, MA, United States). The OPA1 antibodies (ab42364) were purchased from Abcam (Cambridge, MA, United States). The antibodies against Mfn1 (sc-166644), Mfn2 (sc-100560), PINK1 (sc-33796), and DRP-1 (sc-271583) as well as the horseradish peroxidase-conjugated secondary antibodies (sc-2004, sc-2005, sc-2020, and sc-2438) were obtained from Santa Cruz Biotechnology.

To identify the field analyzed in the functional (FM1–43) experiments, the chambers subjected to post hoc immunohistochemistry were marked to ensure their position. After FM1–43 unloading, cultured neurons were fixed with 4% ice-cold paraformaldehyde for 30 min and then permeabilized with PBS containing 0.1% Triton and 5% goat serum for 1 h at room temperature, followed by incubation with primary antibody: mouse anti-MAP2 (1:5000, sc-33796, Santa Cruz Biotechnology), followed by the conjugation of a goat anti-mouse antibody.

Brain slices from the indicated Tg mice were subjected to double immunostaining with rabbit anti-EP (Cat# 36-3000, Invitrogen) and mouse anti-MAP2 (1:5000, sc-33796, Santa Cruz Biotechnology) at 4 °C overnight, followed by the conjugation of goat anti-rabbit Alexa Fluor488 and goat anti-mouse Alexa Fluor594. The staining images were taken under a Leica confocal microscope and analyzed by Universal Metamorp Image Program.

Brain slices from the indicated Tg mice were co‐immunostained with primary antibodies: rabbit anti‐PITRM1 IgG antibody (1:2000), mouse anti‐Cytochrome C Oxidase (CCO, 1: 5000), PSD‐95 (ab16495, Abcam, 1:3000), synapsin 1 (s8067, sigma 1:5000), mouse anti‐MAP2 (1:5000, sc‐33796, Santa Cruz Biotechnology), rat anti‐CD11b (550282, BD Pharmingen, 1:1000), and mouse anti‐GFAP (Glial Fibrillary Acidic Protein, 3670, Cell Signaling, 1:3000) at 4°C overnight. Sections were then incubated with Alexa Fluor 488‐conjugated goat anti‐rabbit IgG/ 594 goat anti‐mouse or rat IgG or Alexa Fluor 594‐conjugated goat anti‐rabbit IgG/488 goat anti‐mouse IgG secondary antibodies, respectively, for 1 h at room temperature. Nuclei were stained by DRAQ5 (5 μM, Cell Signaling) for 10 min at room temperature. Images were taken using a Leica LS5 Confocal Microscope and analyzed using Universal Metamorph Image Program.

Mouse PTECs were lysed in radioimmunoprecipitation assay buffer (RIPA, Pierce Biotechnology, Rockford, IL, USA) and then processed as described previously^{3 (link)}. Briefly, supernatants were retained and assayed for protein content by the Bradford method. Samples (30 μg) were separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and were electroblotted onto a polyvinylidene difluoride membrane. After blocking, membranes were incubated with primary anti-P62 (1:300), anti-P16 (1:500), anti-P21 (1:500), anti-PINK1 (1:300, sc-33796, Santa Cruz Biotechnology), anti-Parkin (1:300), anti-TOMM20 (1:300), anti-α-tubulin (1:500 dilution, sc-398103, Santa Cruz Biotechnology), anti-NDP52 (1:500 dilution, sc-376540, Santa Cruz Biotechnology), anti-NDP52 (1:500 dilution, sc-376540, Santa Cruz Biotechnology), anti-DFCP1 (1:500 dilution, ab90029, Abcam), anti-WIPI1 (1:1000 dilution, ab128901, Abcam), or anti-β-actin (1:1000 dilution, ab8226, Abcam) overnight at 4 °C. Membranes were then washed and incubated with secondary HRP-conjugated antibodies (1:2000) for 1 h at room temperature. Immunoreactive bands were detected by using the enhanced chemiluminescence system (Amersham Biosciences, UK) and a Bio-Rad electrophoresis image analyzer (Bio-Rad, Hemel Hampstead, UK).