Bicinchoninic acid (bca)

The substantia nigra of rat brains was homogenized at 1:10 (w/v) in the homogenate buffer supplemented with a protease inhibitor cocktail. After centrifugation (12,000 g, 10 min), the protein lysate was determined using the bicinchoninic acid (Bio-Rad, Hercules, CA, USA). Protein isolation was performed by 10% SDS-PAGE (Beyotime, Shanghai, China) and moved to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA). Following 2-h blockade with PBS buffer + 5% skim milk powder, membranes were probed overnight with primary antibodies at 4°C, and with the secondary antibody IgG (1:5000, ab205718, Abcam, UK) for 2 h. Enhanced chemiluminescence kits were adopted to visualize the protein bands, and ImageJ software was employed for quantitative analyses. The primary antibodies were LC3 (1:2000, ab192890, Abcam), P62 (1:1000, ab109012), SIRT1 (1:1000, ab189494), AMPK (1:1000, ab32047), phospho-AMPK (p-AMPK, 1:500, ab131357), mTOR (1:10000, ab134903), phospho-mTOR (p-mTOR, 1:1000, ab137133), and GAPDH (1:10000, ab181603). GAPDH was employed as an internal reference.

E. coli ATCC 2592 was cultured in Luria-Bertani broth medium at 37°C overnight. The cultured bacteria were resuspended in 1× phosphate-buffered saline (PBS), lysed using a probe sonicator, followed by centrifugation at 100,000 × g at 4°C for 1 h to separate soluble and membrane protein fractions. Soluble fractions were then separated from the membrane pellets. The membrane pellets were washed, followed by resuspension in 1× PBS. The protein concentration was measured by conducting either Bradford assay (Bio-Rad) or bicinchoninic acid (BCA) assay using bovine serum albumin (BSA) as a standard, and lysates were finally diluted to 1 mg/mL protein concentration with 1× PBS, which was used for all further work (22 (link)).

For the Western blot analysis of hypoxia inducible factor-1α (HIF-1α), cells were incubated in 21% or 1% O2 for 72 h. Briefly, samples were collected in RIPA (RadioImmunoPrecipitation Assay) lysis buffer (50 mM Tris Hydrocloride, pH 7.9, 120 mM NaCl, 1% Nonidet, 1 mM EDTA, 5 mM NaF, 1 mM sodium othovanadate, 0.04 mM 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride), and protein concentration was determined by BCA Assay (BicinChoninic Acid)(Bio-Rad, München, Germany). Western blot analysis was performed using the following antibodies: mouse anti-HIF-1α diluted ¹/₅₀₀ (BD Bioscience, San Jose, CA, USA) and rabbit anti-actin diluted ¹/₅₀₀₀ (Santa Cruz, Dallas, TX, USA).

The tissue was homogenized at 1 : 10 (w/v) in homogenate buffer (including 20 mM Tris-HCL, 150 mM Nacl, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM sodium vanadate, 1% Triton X-100 and 1 mg/mL leupeptin hemisulfate salt; pH 7.5) and added with protease inhibitor cocktail. After the homogenate was centrifuged at 12000 g for 10 min, the protein lysate was quantified using the bicinchoninic acid (Bio-Rad, Hercules, CA, USA) method. Protein was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime, Shanghai, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After sealing in PBS buffer containing 5% skim milk powder at room temperature for 2 h, the membranes were incubated with primary antibodies at 4°C overnight, and then the membranes were incubated with the secondary antibody IgG (1 : 5000, ab205718, Abcam, UK) at room temperature for 2 h. The enhanced chemiluminescence kit (Thermo Fisher Scientific) was used to detect the protein bands and Image J software was used for quantitative analysis. The primary antibodies included LC3 (1 : 2000, ab192890, Abcam), P62 (1 : 1000, ab109012, Abcam), NIX (1 : 1000, ab109414, Abcam), BNIP3 (1 : 1000, ab109362, Abcam), and β-actin (1 : 1000, ab8227, Abcam).

B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) protein concentrations were first determined using a bicinchoninic acid (BCA; Bio-Rad, Hercules, CA, USA) protein assay kit following the manufacturers’ instructions. Proteins were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel, transferred to a nitrocellulose membrane, blocked for 30 min at 37°C with 5% skimmed dry milk and incubated with a primary antibody overnight on a rocking platform. The primary antibodies used were the following: Mouse monoclonal against TLR4 (ABCAm), rabbit against NFκB p50, rabbit against Bcl-2 and rabbit against Bax (all from Santa Cruz Biotechnology, Santa Cruz, CA, USA). Membranes were subsequently washed three times with Tris-buffered saline and Tween 20 (TBST) and incubated with the secondary antibody in TBST solution for 30 min at 37°C after which they were washed as above. The secondary antibodies used were the following: Peroxidase-labeled goat anti-rabbit IgG and peroxidase-labeled rabbit anti-mouse IgG (both from Zhongshan Company, Beijing, China). Immunoblots were developed using an enhanced chemiluminescent reagent kit (ABCAm, Cambridge, UK). The bands were scanned and quantified by densitometric analysis using an image analyzer (Tanon2500, Shanghai, China).