Enhanced chemiluminescence agent

Proteins were extracted from kidney tissues or HK-2 cells using RIPA buffer containing 4% cocktail proteinase inhibitors and then analyzed by western blotting. Equal amounts of protein were separated by SDS-polycrylamide gels and then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies against FABP4 (Abcam, Cambridge, MA, USA), Bax (Abcam, Cambridge, MA, USA), Bcl-2 (Abcam, Cambridge, MA, USA), cleaved-casp-3 (Cell Signaling Technology), GRP78 (Cell Signaling Technology), CHOP (Cell Signaling Technology), caspase-12 (Cell Signaling Technology, Beverly, MA, USA) and β-actin (Abcam, Cambridge, MA, USA) overnight at 4 °C followed by incubation with secondary antibodies (R&D Systems, MI, USA) for 1 h at room temperature. Finally, the proteins were developed with an enhanced chemiluminescence agent (Millipore Corporation, Boston, MA, USA). The signals were measured using an Odyssey Infrared Imaging System (Bio-Rad, ChemiDoc MP, mANUSC, Bio-Rad Laboratories Inc., Hercules, CA, USA) and quantified using the Image J program (National Institutes of Health, Bethesda, MD, USA). The ratio for the target proteins were normalized against β-actin.

Cells were lysed with RIPA lysis buffer and centrifuged at 12,000 rpm at 4°C. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay kit (Thermo, Rockford, USA). The samples corresponding to 20 μg of protein were resolved on a 10% denatured sodium dodecyl sulfate (SDS) polyacrylamide gel, and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking non-specific binding sites for 1 h with 5% skim milk, the membranes were incubated with PBX3 antibodies (1: 2000 dilution; Abcam, Cambridge, UK) overnight at 4°C. Then the membranes were washed with Tris-buffered saline (TBST) and incubated with a secondary antibody for 1 h. The protein bands were detected by enhanced chemiluminescence agent (Millipore, Billerica, MA, USA). Antibody of β-Actin (1:5000 dilution; Cell Signal Technology, Beverly, MA, USA) was used as the loading control.

Proteins were isolated from renal tissues or HK-2 cells with a RIPA lysis buffer and then analyzed by Western blotting. In brief, equal amounts of protein were separated by SDS-PAGE and then transferred on to a PVDF membrane (Bio-Rad, Hercules, CA, USA). Subsequently, the membranes were incubated with primary antibodies against NF-κB p65 (Abcam, Cambridge, MA, USA), TLR4 (Abcam, Cambridge, MA, USA), and GAPDH (Origene, Rockville, MD, USA) overnight at 4 °C. Then the membranes were incubated with HRP-conjugated secondary antibodies (R&D Systems, Minneapolis, MN, USA) for 1 h at room temperature. Finally, the proteins on the membrane were developed with an enhanced chemiluminescence agent (Millipore Corporation, Boston, MA, USA). The signals were detected using an Odyssey Infrared Imaging System (Bio-Rad, ChemiDoc MP, mANUSC, Bio-Rad Laboratories Inc., Hercules, CA, USA) and analyzed with the Image J program (National Institutes of Health, Bethesda, MD, USA). The ratio for the protein was normalized against GAPDH.

After washed with ice cold D-hank’s, astrocytes were lysed in RIPA buffer. Protein concentration was determined using the BCA Bradford protein assay kit, and protein samples were mixed with SDS-PAGE loading buffer. After heated at 100°C for 8 min, proteins were subjected to 10–12% SDS-PAGE gel and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, USA) and then blocked with Tris-buffered saline including 5% nonfat milk and 0.1% Tween-20 for 2 h at room temperature. Incubation with primary antibody against NF-κBp65 (1:1000, CST, USA), p-IκBα antibody (1:1000, CST, USA), p-IKK (1:1000, CST, USA), iNOS (1:1000, Abcam, USA), IL-1β (1:1000, Abcam, USA), ICAM-1 (1:1000, Abcam, USA), VCAM-1 (1:1000, Abcam, USA), β-actin (1:4,000, CST, USA), and LaminB1 (1:1000, Abcam, USA) was done overnight at 4°C and then with HRP-conjugated secondary antibody (1:2000). Antibody positive bands were detected with an enhanced chemiluminescence agent (Millipore, USA). Quantification of bands was made by scanned densitometric analysis and Quantity one analysis system.

After treatment, neurons were washed with ice cold D-hank's and lysed in RIPA buffer. Protein concentration was measured using the BCA Bradford protein assay kit and mixed with SDS-PAGE loading buffer. Following heating at 100°C for 8 min, proteins were subjected to 10–12% SDS-PAGE gel and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, USA) and then blocked with 5% nonfat milk in PBS-T (PBS containing 0.1% Triton X-100) for 2 hours at room temperature. Incubation with primary antibody against Bcl-2 (1 : 1000), Bax (1 : 1000), cleaved caspase-3 (1 : 1000), p-Akt (1 : 1000), and β-actin (1 : 1000) was done overnight at 4°C and then with HRP-conjugated secondary antibody (1 : 2000). Antibody positive bands were detected with an enhanced chemiluminescence agent (Millipore, USA). The band densities were quantified by Image J software.

The total proteins extracted from cells with RIPA lysis buffer (Beyotime, China) were quantified using a BCA Protein Assay Kit (Beyotime). A total of 50 µg of protein was separated by 10% of SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Bio‑Rad Laboratories, Inc.). The membrane was then blocked using 5% of skim milk and incubated with SSAO (1:1,000; #PA5-104398; Gibco), iNOS (1:1,000; #PA5-37667; Gibco), ROCK1 (1:1,000; #PA5-22262; Gibco), MLC₂₀ (1:1,000; #PA5-17727; Gibco), and GAPDH (1:500; #PA1-987-HRP; Gibco) antibodies overnight at 4°C. After washing three times in TBST, the membranes were incubated with anti-Rabbit IgG (H + L) HRP at a dilution of 1:3,000 in TBST containing 5% of skim milk for 2 h at 37°C. The protein bands were detected by enhanced chemiluminescence agent (Millipore, Billerica, MA, USA).

Proteins (Supplementary Table S1) were extracted from kidney tissues or HK-2 cells using RIPA buffer containing 4% cocktail proteinase inhibitors and then analyzed by western blotting. Equal amounts of protein were separated by SDS-polyacrylamide gels and then transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, United States). The membranes were incubated with primary antibodies against overnight at 4°C followed by incubation with secondary antibodies (R&D Systems, MI, United States) for 1 h at room temperature. Finally, the proteins were developed with an enhanced chemiluminescence agent (Millipore Corporation, Boston, MA, United States). The signals were measured using an Odyssey Infrared Imaging System (Bio-Rad, ChemiDoc MP, mANUSC, Bio-Rad Laboratories Inc., Hercules, CA, United States) and quantified using the ImageJ program (National Institutes of Health, Bethesda, MD, United States).