Anti tom20 sc 11415

The protein cytochrome c, Bcl-2 and Bax were assessed in the cytosolic and/or mitochondrial fractions of cortical neurons extracts in the presence/absence of AMD3100 treatment (200 nM, 24 h). Equal amounts of protein in a volume of 30 µL were separated on a 15% polyacrylamide gel (SDS-PAGE). These gels were transferred to a polyvinylidene fluoride (PVDF) membrane (GE Healthcare, Madrid, Spain). All membranes were blocked by adding 5% nonfat dry milk for 1 h at room temperature. The membranes were incubated overnight at 4 °C with the appropriate primary antibodies: goat polyclonal anti-cytochrome c for immunodetection (1/500) (sc-7159, Santa Cruz Biotechnology, Quimigen, Madrid, Spain), rabbit polyclonal anti-Bax (1/250) (sc-493), and anti-Bcl-2 (1/250) (sc-492) (Santa Cruz Biotechnology, Quimigen, Madrid, Spain) followed by incubation with peroxide-conjugated secondary antibodies for 1 h at room temperature (Santa Cruz Biotechnology, Quimigen, Madrid, Spain). Blots were developed by enhanced chemiluminescence (ECL select; GE Healthcare, Madrid, Spain) following manufacturer’s instructions. Anti β-actin (sc-47778) and anti-Tom-20 (sc-11415) antibodies (Santa Cruz Biotechnology, Quimigen, Madrid, Spain) were used as loading control for cytosolic and mitochondrial extracts, respectively.

Cells were lysed in RIPA buffer and subjected to western blot analysis as described previously 51 (link). Anti-Cox2 (ab198286), anti-MFN2 (ab56889), anti-SDHB (ab14714), anti-P4HB (ab137110), anti-PMP70 (ab3421), anti-PGC-1α (ab191838), anti-TFAM (ab272885), and anti-NRF1 (ab175932) antibodies were purchased from Abcam (Cambridge, UK). An anti-LC3B (#L10382) antibody was purchased from Invitrogen (Waltham, MA, USA). Anti-ULK1 pS555 (#5869), anti-ULK1 pS317 (#37762), anti-ULK1 (#8054), and anti-GM130 (#12480) antibodies were purchased from Cell Signaling Technology (Danvers MA, USA). An anti-OPA1 (#612606) antibody was purchased from BD Biosciences (San Jose, CA, USA). An anti-PINK1 (#BC100-494) antibody was purchased from Novus Biologicals (Littleton, CO, USA). Anti-Tom20 (SC-11415) and anti-Actin (SC-47778) antibodies were purchased from Santa Cruz (Dallas, TX, USA). All western blot analyses were repeated three times. Band intensities were quantified using densitometry and ImageJ software (NIH).

GCs were grown on coverslips in 12-well plates for 4 days and then exposed to the treatments described above. After washing in PBS, the cell climbing sheets were fixed with 4% paraformaldehyde (PFA) for 1 h, permeabilized using 0.5% Triton X-100 in PBS for 10 min at 4 °C, and blocked with 1% BSA for 1 h at room temperature. The coverslips were then incubated with 1:100 dilutions of anti-Tom20 (sc-11415; Santa Cruz), anti-Parkin (sc-32282; Santa Cruz), or anti-LC3 (L7543; Sigma) for 1 h at 37 °C. They were subsequently stained for another 1 h with rabbit or mouse Alexa Fluor 488 (A-11008), 568 (A-11031), and 633 (A-21072) secondary antibodies at 1:200 dilutions (Invitrogen). The coverslips were washed, mounted on slides, and observed under a laser-scanning confocal microscope (Zeiss LSM 710 META).

Total cell lysates were prepared by lysing cells in RIPA lysis buffer for 15 min on ice and removal of insoluble material by centrifugation at 16000g for 10 min at 4˚C. Protein content was measured using the Bio-Rad protein assay and equal protein amounts in 1x Laemmli buffer were used for SDS-PAGE. For immunoblotting, proteins were transferred electrophoretically to nitrocellulose membranes and exposed to the following primary antibodies: anti-MPC1 (HPA045119, Sigma), anti-MPC2 (home-made), anti-TOM20 (sc-11415, Santa-Cruz), anti β-tubulin (T4026, Sigma). Images of Western blotting were uniformly treated for contrast enhancement using Adobe Photoshop.