Bicinchoninic acid kit

All reference standard chemicals were obtained from National Institutes for Food and Drug Control, China¹, including berberine hydrochloride (C₂₀H₁₈ClNO₄, PubChem CID: 12456, Lot No.: 111895–201504), mangiferin (C₁₉ H₁₈O₁₁, PubChem CID: 5281647, Lot No.: 111607–201503), and phellodendrine chloride (C₂₀H₂₄ClNO₄, PubChem CID: 59818, Lot No.: 110713–201212). Lipofectamine 2000 and MitoTracker Green (MTG) were purchased from Invitrogen (Grand Island, NY, United States). 1-methyl-4-phenylpyridinium (MPP⁺) were obtained from Sigma-Aldrich (St. Louis, MO, United States). The bicinchoninic acid kit, protease and phosphatase inhibitors, and enhanced chemiluminescence kit were bought from Applygen (Beijing, China). The used antibodies as the following: rabbit anti-DJ-1, rabbit anti-PI3K, rabbit anti-Akt, rabbit anti-Akt phosphorylation^Thr308, rabbit anti-Akt phosphorylation^Ser473 were obtained from Cell Signaling (Beverly, MA, United States). Mouse anti-beta-action primary antibody and all secondary antibodies were obtained from Zhong-Shan (Beijing, China).

Heart tissues obtained from 8-week C57BL/6 mice were lysed in RIPA cell lysate buffer supplemented with protease (Roche Diagnostics) and phosphatase inhibitors (Roche Diagnostics); tissue lysates were subsequently centrifuged at 12,000 rpm for 10 min at 4°C. A bicinchoninic acid kit (Applygen Technologies, Inc.) was used to assess the protein concentration. Polyvinylidene fluoride membranes were incubated with the following primary antibodies overnight at 4°C: anti-TECRL (1:1,000; Aviva System Biology, Inc.) and anti-Tubulin (1:1,000; Cell Signaling Technology, Inc.). The secondary antibody (1:2,000; Cell Signaling Technology, Inc.) was diluted and incubated for 1 h at room temperature. An immunology scanner (GS-800, Bio-Rad Laboratories, Inc.) was used to measure the densities of the protein bands following exposure of the membranes to a chemiluminescent substrate (ECL, PerkinElmer, Inc.).

Protein concentration was assessed using a bicinchoninic acid kit (Applygen Technologies Inc., Beijing, China) according to the method described by Li et al [15 (link)]. Western blot was performed according to our previous report [16 (link)]. Briefly, the protein sample was combined with the loading buffer, then heated in a 95°C water bath for 10 min. Protein samples (25 μg) were separated by 10% sodium dodecyl sulfate-polyacryl amide gel electrophoresis protein over 2 h at 80 v, and transferred to 0.45 μm polyvinylidene fluoride membranes (Millipore Corp., Billerica, MA, USA). After blocking with 5% (w/v) skim milk, the membranes were incubated overnight at 4°C with the primary antibodies: PEPCK1 (1:2,000; Bioss Biotechnology Co., Ltd, Beijing, China), PEPCK2 (1:2,000; Bioss, China), G6PC (1:2,000, Bioss, China), PC (1:1,000, Ab-mart, Biomedical Co., Ltd, Shanghai, China), and β-actin (Bioss, China). After washing for 5 min in Tris buffered saline with Tween 20, the membranes were incubated goat anti-rabbit secondary antibodies (Sangon, Shanghai, China) for 1 h. Finally, the reaction strips were evaluated using Bio-Rad software (version 5.2.1). The gray values of the target proteins and the β-actin were analyzed using Image J software. Then the gray values of the target protein were divided by the gray values of the β-actin and normalized.