Odyssey fc system

Whole cell lysate samples were prepared using Laemmli buffer, resolved in precast SDS-PAGE gels (Life Technologies), and transferred onto Immobilon-PSQ Membranes (Millipore). The membranes were incubated in buffers from the WesternBreeze Blocker/Diluent kit (Life Technologies) and probed with corresponding primary antibodies (S3 Table). Bound antibodies were detected by IRDye secondary antibodies using the Li-COR Odyssey Fc system (Li-COR).

Cells were lysed in lysis buffer (SDS 1%, Tris pH 7.4 10 mM, Sodium orthovanadate 1 mM). Samples in Laemmli buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, 0.001% bromophenol blue) containing equal amounts of total protein extracts were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to PVDF membranes (Thermo Scientific, Waltham, MA, USA), membranes were probed with primary antibodies PARP, β-tubulin (Cell signaling, Leiden, The Netherland) or ubiquitin (Abcam, Paris, France). Membranes were then probed with secondary fluorescent antibodies (Li-COR IRDye). Antibody binding was visualized with the LI-COR odyssey Fc system (Cambridge, UK).

Renal tissues or HK-2 cell samples were lysed using radioimmunoprecipitation assay (RIPA) buffer. Mitochondrial proteins were extracted using a Mitochondrial/Cytoplasmic Protein Extraction Kit (Beyotime, Shanghai, China), according to the manufacturer’s instructions. Equal amounts of protein were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, United States). The membrane was incubated with primary antibodies specific to KIM1 (1:1000 dilution, Novus), α-SMA, E-cadherin, TGFB1 (1:1000 dilution, Abcam), carnitine palmitoyltransferase 1A (CPT1), acyl-CoA oxidase 1 (ACOX1), cytochrome C (CYCS), AMPK and phosphorylated (p)-AMPK (1:1000 dilution, Cell Signaling Technology, Danvers, MA, United States), collagen I, and β-actin (1:1000 dilution, Proteintech) overnight at 4°C. The membranes were then incubated with goat anti-rabbit or mouse IgG horseradish peroxidase conjugate (1:10,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, United States) and scanned using an Odyssey Fc System (LI-COR, Lincoln, NE, United States).

The cells were incubated with RIPA buffer containing protease inhibitors (protease inhibitors: RIPA = 1:100, Solarbio, Guangzhou, China) for 15 min on ice to fully lyse the cells 48 h after transfection. Then, the cells were centrifuged at 12,000× g for 10 min at 4 °C, and the supernatant was removed. Total protein was then quantified using the BCA Protein Assay Kit (Beyotime, Shanghai, China). Proteins were separated in 12% SDS-PAGE and transferred to nitrocellulose membranes (Whatman, Maidstone, UK), blocked with 5% skim milk powder for 1 h, and then incubated with primary antibody solution overnight at 4 °C. Subsequently, PVDF membranes were washed three times for 5 min with TBST solution (Beyotime) and then incubated with secondary antibody solution for 60 min at room temperature. Western immunoblotting results were analyzed using the Odyssey Fc system (LI-COR, Lincoln, NE, USA). Antibody information is as follows: actived-caspase-3 p17 polyclonal antibody (BS7004; Bioworld, MN, USA; 1: 500) cleaved caspase-8 (Asp391) (18C8) rabbit mAb (9496; Cell Signaling Technology, Danvers, MA, USA; 1:1000), anti-caspase-9 antibody [E23] (ab32539; Abcam, Cambridge, UK; 1:1000), rabbit anti-GAPDH (AB-P-R 001; Hangzhou Goodhere Biotechnology Co., Ltd., Hangzhou, China; 1:1500).

Two million BMMCs were pelleted by centrifugation and lysed in SDS lysis buffer (40 mm Tris‐HCl (pH 7.5), 0.8 mm EGTA, 0.8 mm EDTA, 0.8 mm sodium orthovanadate, 40 mm sodium fluoride, 0.8 mm sodium pyrophosphate, 0.22 m sucrose, 1% (v/v) Triton X‐100, 0.1% (v/v) 2‐mercaptoethanol, 2% (w/v) SDS, and 10% glycerol). Lysates were heated at 95°C for 5 min and DNA sheared by passing the lysates through a 25-gauge needle. Samples were run on 10% SDS tris/glycine polyacrylamide gels and transferred on to nitrocellulose membranes using standard techniques. Primary antibodies were from Cell Signalling Technologies. Horse radish peroxidase-conjugated secondary antibodies were from Thermo Fisher Scientific. Detection was carried out using Clarity ECL reagent (Bio-Rad) and imaged on an Odyssey Fc system (Licor) and analysed using Image Studio software. Full membrane images of the blots are shown in Supplementary Figure S1.