Rabbit anti tfam

Cell lysates were prepared with lysis buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 μg/ml leupeptin. 1 mM PMSF was added immediately prior to use. The protein concentration was measured by DC protein assay (Bio-Rad, Irvine, CA). Quick neuron nuclear extract preparation: the cells were rinsed with PBS once, and plates placed on ice and 1 ml of ice cold Buffer A (25 mM Hepes pH 7.0, 25 mM KCl, 0.05 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.1% NP-40, 1 mM DTT) added. The plate was scraped and cells were transferred into an Eppendorf tube, which was centrifuged and rinsed once with Buffer A (no NP-40). The pellet was resuspended in PBS, and 2x SDS PAGE sample buffer added, prior to boiling for 10 min at 95°C. The following primary antibodies and dilutions were used: antibodies against the major glycolysis enzymes were from Cell Signaling sold as glycolysis antibody sampler kits (#8337&12866); all were used at 1:1000; Rabbit anti-TFAM (Cell Signaling) used at 1:1000; Rabbit anti-CS, IDH2, PGC-1α and ATP5O (Abcam, Cambridge, United Kingdom) used at 1:1000, and mouse anti-Sucla2 and goat anti-Hsp60 (Santa Cruz Biotechnology) used at 1:1000. Immunoblotting results were analyzed by Odyssey Imager (Licor, Lincoln, NE) scanning.

The different cell types were seeded in 24-well plates containing poly-D-lysine (20 µg/mL) pretreated glass coverslips. After treatments, they were fixed for 20 min with 4% paraformaldehyde and blocked for 30 min at 37 °C with 5% goat serum in a solution of 1% Triton X-100 in PB. After that, cells were incubated for 1 h at 37 °C with different primary antibodies: mouse anti-C/EBPβ (C19 clone, Santa Cruz, ref. sc7962), rabbit anti-TFAM (Cell Signaling, Leiden, The Netherlands, ref. 8076), or rabbit anti-p62 (Sigma, ref. P00067). Cover-slips were then washed with PB and incubated for 45 min at 37 °C with Alexa-labeled secondary antibodies (Thermo Fisher, Madrid, Spain). Staining of nuclei was performed using 4′,6-Diamidino-2-phenylindole (DAPI). Images were acquired in a LSM710 laser scanning spectral confocal microscope (Zeiss) and the microscope settings were adjusted to produce the optimum signal-to-noise ratio. Quantitative analysis of fluorescence intensity and particles number was performed by the use of Image-J software, and at least five different counting fields were randomly selected from three independent experiments.