Nupage bis tris mini gel

Cells were lysed in RIPA cell lysis buffer containing protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Lysates were sonicated for 1 min and centrifuged at 14, 000 × g for 10 min at 4 °C. Twenty micrograms of proteins were resolved in 4–10% NuPAGE Bis-Tris Mini gels (Invitrogen) and electrophoretically transferred to a nitrocellulose membrane (Invitrogen). The unspecific binding sites were blocked in blocking solution containing 5% milk or bovine serum albumin (BSA) for 1 h at room temperature. Then the membranes were incubated overnight in primary antibodies followed by incubation in secondary antibodies for 1 h at room temperature. The reaction products in the membranes were visualized using an enhanced chemiluminescence Western Blot Detection Kit (Thermo Scientific). For immunoprecipitation experiments, 500 µg of total protein lysates were incubated overnight at 4°C with anti-Flag agarose beads (Santa Cruz). The primary antibodies those against NF-κB p65 (Thermo Scientific); p300 (Abcam); SOD2 (Santa Cruz); total OXPHOS (Abcam) and β-actin (Sigma-Aldrich). The densities of protein bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD) and normalized to the β-actin band intensity for the same sample.

CD-1 mouse embryos at E13.5 (n=4), E16.5 (n=6) or new-born mice (n=6) were decapitated and forebrains were removed. Crudely purified mitochondrial fractions were prepared as described (Morozov et al., 2013 (link)). 20 μg protein samples were separated using electrophoresis at 210V in 4-12% NuPAGE Bis-Tris mini gels (Invitrogen, Carlsbad, CA, USA) and electrophoretically transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membranes were subsequently immunoblotted with anti-CB₁-L31 (made-in-Guinea pig; Frontier Science Co. Ltd, Japan; 1:400) and anti-SLP2 (made-in-rabbit; 1:200; Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA). The membranes were counterstained using corresponding donkey anti-Guinea pig (1:5000; Jackson Immunoresearch, West Grove, PA, USA) or goat anti-rabbit (1:3000; Bio-Rad Laboratories, Hercules, CA, USA) horse radish peroxidase conjugates, immersed in Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) and exposed to X-ray films during recorded periods. Between the immunoblot procedures, membranes were rinsed and incubated in Restore Western Blot Stripping Buffer (Thermo Scientific; Rockford, IL, USA). Before repetitive immunoblots, the membranes were kept in Tris-saline buffer (pH 7.5) at +4°C for 4 days.