Ecl detection kit

To extract the proteins from the tissues, 3 IMN kidney tissues and 3 NATs were cut into pieces and lysed on ice using RIPA lysis buffer (Applygen, China) supplemented with Protease Inhibitor Cocktail, and then quantified using the BCA Protein Assay Kit. According to the expression of the internal reference protein GAPDH in the specimen, 90 µg extracted proteins from IMN kidney tissue and 10 µg extracted proteins NATs were separated by 12 % SDS-PAGE and transferred to a nitrocellulose (NC) membrane (Millipore, Darmstadt, Germany). After blocking with 5 % non-fat dry milk in tris-buffered saline and 0.1 % Tween 20 solution, membranes were incubated overnight at 4 ℃ with primary antibodies against the following proteins: MMP9 (ABGENT, China, 1:1000), CAPN1 (Abclonal, China, 1:1000), MMP9, MMP14, CTSS, and GAPDH (Proteintech, China, 1:5000). The membranes were then incubated with the appropriate HRP-Goat Anti-Rabbit (Elabscience, China, 1:2,500) and HRP-Sheep Anti-Mouse (Jackson ImmunoResearch, USA, 1:8,000) secondary antibody for 1 h at room temperature. The specific bands were detected using an ECL detection kit (Applygen, China) and captured on an ImageQuant LAS 4000 mini system (GE Healthcare, NJ, USA). Relative expression was determined by normalizing to GAPDH expression using the ImageJ software (Version 1.51j, National Institutes of Health, MD, USA).

Western blot analysis was used to detect protein expression. Total protein was extracted from mouse livers using RIPA lysis buffer (Applygen Technologies, Beijing, China), and protein concentrations were determined using a BCA protein assay kit (Applygen Technologies, Beijing, China). Thirty micrograms of total protein per lane were separated on 10% SDS–PAGE gels and transferred onto PVDF membranes (Roche, Indianapolis, IN, United States). The membranes were blocked with TBST containing 3% nonfat milk for 2 h at room temperature and then incubated with primary antibodies overnight at 4°C. The primary antibodies against Acc, β-actin, Fas, Scd1, BCKDH-E1α, p-BCKDH-E1α, and Atgl were purchased from Cell Signaling Technology (Beverly, MA, United States). The primary antibody against Cpt1A was purchased from Abcam (Cambridge, MA, United States). After binding with secondary antibodies for 2 h at room temperature, the bands were detected using an ECL detection kit (Applygen Technologies, Beijing, China). Band intensity was assessed by densitometry and expressed as the mean density area using ImageJ analysis software.

Antibodies against IkB-α (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha; 1:1,000), JNK (c-Jun N-terminal), phosphorylated JNK (1:1,000), β-actin (1:1,000) and TLR4 (1:1,000) were purchased from Cell Signaling Technology (United States). Total protein of liver and adipose tissue was extracted by adding lytic buffer to each ice-cold sample. Proteins were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millikon). Each membrane was sealed in the presence of Tris buffered saline containing Tween (TBST) containing 5% skimmed milk powder at room temperature for 1 h. Then, a defined concentration of diluted antibody was added as described by the manufacturer. The blot was incubated overnight in a flip shaking bed at 4 °C and washed six times for 5 min each time using TBST. TBST containing 5% skimmed milk powder a 1:8,000 dilution of mouse anti-rabbit antibody labeled with horseradish peroxidase (HRP) and incubated at room temperature for 2 h. The blot was washed six times for 5 min each time. An enhanced chemiluminescence (ECL) detection kit (Applygen Technologies, Beijing, China) was used to display protein bands. The protein bands were analyzed by ImageJ image analysis software (NIH, Bethesda, MD, United States).