Bca protein assay

The brain tissues were washed twice with cold PBS and lysed in extraction buffer (20 mM HEPES [pH 7.4], 2 mM EDTA, 50 mM glycerophosphate, 1 mM dithiothreitol, 1 mM Na₃VO₄, 1% Triton X-100, and 10% glycerol) on ice. The lysates were centrifuged at 12,000 rpm for 15 min. Supernatants were collected, and protein concentrations were determined by bicinchoninic (BCA) Protein Assay (Bio-Rad, Hercules, CA). Proteins were boiled in sample buffer, separated in 12% sodium dodecyl sulfate-polyacrylamide gels by electrophoresis (50 μg/lane), and electroblotted onto nitrocellulose membranes (Pall Corporation, Ann Arbor, MI). Transferred blots were incubated sequentially with blocking agent (5% non-fat milk in Tris-buffered saline), anti-iNOS antibody (1:125 dilution, ab15323, Abcam), anti-Arg-1 antibody (1:500 dilution, ab91279, Abcam), and secondary antibody blots prior to development with enhanced chemifluorescence detection kits and exposure Hyperfilm (Fuji, Tokyo, Japan) according to the manufacturer's directions. The same blots were subsequently stripped and reblotted with antibodies that recognized β-actin (Sigma) as a reference to verify equal amounts of the protein among samples. Graphs of blots were obtained in the linear range of detection and were quantified for the level of specific induction with the Scion Image System.

Young adult worms were collected by washing them off two 6-cm plates. Worms were washed with M9 buffer before being frozen in 100 μl of ultrapure water at −80°C overnight. The next day, frozen worms were immersed in boiling water for 20 min to release ATP and destroy ATPase activity. The samples were placed on ice for 5 min before being centrifuged at maximum speed at 4°C. The supernatant was collected, and 5 or 10 μl of the supernatant was used to determine ATP levels using the ATP Bioluminescence Assay Kit CLS II (Sigma-Aldrich). Samples were loaded into a 96-well white opaque polystyrene plate (Pierce), and luminescence was measured in a plate reader (TECAN, Infinite M1000). ATP levels were normalized to protein levels. Protein levels were determined using 12.5 μl of the supernatant in a BCA protein assay (Bio-Rad). ATP levels in Fig. 5H were normalized to the untreated WT level measured on the same day, which averaged 26.46 pmol/μg protein.

A western blotting assay was performed to detect the protein level of MEKK3 and p-p65 in cultured cells and selected mouse midbrains. Total protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B; Beyotime, Jiangsu, China) with protease and phosphatase inhibitors (B14001 and B15001; BioTools, Olathe, KS, USA), following the manufacturer’s protocol. The protein concentration was measured with bicinchoninic acid (BCA) protein assay (Bio-Rad Laboratories, Inc., Berkeley, CA, USA). Equal amounts of protein were isolated using sodium dodecyl sulphate polyacrylamide gel electrophoresis (Beyotime Biotechnology, Shanghai, China) and then transferred to a polyvinylidene fluoride membrane (IPVH00010; Millipore, Bedford, USA). After blocking in 5% Tris-buffered saline-Tween, the membrane was incubated with primary antibody at 4 °C overnight. The antibodies used were as follows: rabbit anti-MEKK3 (NB100-92399; Novus, USA), mouse anti-p-p65 (Ser536; Cell Signaling Technology, USA), rabbit anti-GAPDH (ab8245; Abcam, Cambridge, MA, USA), goat anti-rabbit IgG-HRP (31460; Life Technologies, USA), and rabbit anti-mouse IgG (A-21065; Life Technologies).