Reinforced supported nitrocellulose membranes

Neurons were washed once with warm 1X phosphate-buffered saline (PBS) and lysed using TNN lysis buffer (40 mM Tris-HCL pH 7.4, 150 mM NaCl, 1 mM DTT, 1 mM EDTA, 1% NP40, and 1% protease/phosphatase inhibitor cocktail). Protein extracts were eluted with Laemmli sample buffer, heated at 95°C for 10 min, resolved by SDS–PAGE and transferred to reinforced supported nitrocellulose membranes (Whatman™, Germany) for 2 h at 4°C in a transfer buffer containing 25 mM Tris (pH 7.4), 200 mM glycine, and 20% methanol. Membranes were blocked for 1 h at room temperature with 10% nonfat dry milk in 1X PBS with 0.1% Tween-20 (PBST), washed and incubated with primary antibodies for 2–4 h in 5% nonfat dry milk at room temperature. The blots were subsequently washed three times with 1X PBS and incubated with IRDye® 800CW goat anti-mouse and IRDye® 680RD goat anti-rabbit secondary antibodies and visualized with an Odyssey® CLx Imaging System (LI-COR, Inc., Lincoln, NE). The following antibodies were used for Western blot: Rabbit polyclonal antibodies to LC3 (Sigma, St. Louis MO), GAPDH (Santa Cruz Biotechnologies. Dallas TX), β-tubulin (LI-COR, Lincoln NE), HIV-1 Tat (NIH AIDS Reagents, Germantown MD), Parkin, Tomm20 and PINK1 (Abcam, Cambridge MA). Goat polyclonal antibody to VDAC1 and mouse monoclonal to cytochrome C were from Santa Cruz.