Bradford protein assay

The cells were cultured at a seeding density of 3-5x10⁵ cells in 60-mm plates and maintained at 37°C in 5% CO₂ humidified atmosphere. The cells were irradiated and/or treated with AKBA, then harvested and homogenized in lysis buffer [50 mM Tris–HCl, pH 7.4, 0.5% Triton X-100, 150 mM NaCl, 0.1 mM phenyl-methanesulfonyl-fluoride (PMSF) and complete protease inhibitor cocktail] for 30 min on ice. The lysed cells were centrifuged at 13000 g at 4°C for 15 min and the supernatant was collected. Protein concentration was determined using a Bradford/Bio-Rad Protein Assay (Bio-Rad, Hercules, CA, USA; see below). The supernatants (30–50 mg of protein) were separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel and transferred onto a nitrocellulose membrane. After blocking with 5% non-fat milk in PBS buffer containing 0.1% Tween 20 (PBST), the membranes were incubated for 1 hr at room temperature with primary antibody against PARP (1:1000), β-actin (1:1000) and also incubated with HRP-labeled goat anti-rabbit IgG (1:1000) for 1 hr. All blots were detected using SuperSignal West Pico Chemiluminescent Substrate (ThermoScientific, Waltham, MA, USA) and band density was evaluated using ImageJ software.

Briefly, protein concentrations were measured using Bradford Bio-Rad protein assay according to the manufacturer’s protocol (Bio-Rad, USA). The absorbance measurement was performed following manufacturer’s protocol (Biochrom Asys Expert 96 micro plate reader, Cambridge, UK). 10 μg of proteins were loaded on 4–20% Mini-Protean TGX Precast Gels (Bio-Rad, USA) then transferred to nitrocellulose membranes (Bio-Rad) using Trans-Blot Turbo System (Bio-Rad). Membranes were then blocked with 5% skim milk (Sigma-Aldrich) diluted in PBS-Tween 20 (Sigma-Aldrich). After blocking, the membranes were incubated with the primary antibodies listed in previous section and BMP7 antibody (Abcam; data not shown) at 4 °C, overnight. Incubation with primary monoclonal anti-ß-Actin antibody (1:10,000; Sigma-Aldrich) was performed for 20 minutes only. Membranes were incubated with peroxidase-conjugated secondary antibody (Vector Labs) and blots were developed using Clarity Western ECL Substrate (Bio-Rad) and the protein levels were normalized to actin.

Cells were lysed with RIPA-buffer containing Protease Inhibitor Cocktail (Sigma), incubated for 30 min on ice, centrifuged for 15 min at 13000g. The protein concentration was quantified using Bradford Bio-Rad Protein Assay (BioRad). Total proteins (15 μg) were treated with NuPAGE LDS Sample Buffer (Life Technologies) containing 10% β-mercaptoethanol for 5 min at 96°C, resolved in 10% SDS-PAGE gels and transferred to nitrocellulose membranes Amersham Protran 0.45 NC (Amersham). The membranes were blocked with 5% milk in PBST (0,1%) for 1 h and incubated with primary antibodies [mouse anti-serglycin monoclonal (Santa Cruz, 1:200) or mouse anti-β-Actin monoclonal (Santa Cruz), 1:500] overnight at 4°C followed by secondary goat anti-mouse IgG (H+L)-HRP peroxidase-conjugated antibodies (BioRad) for 2 h at RT. Proteins were detected with an Amersham ECL Western Blotting Detection Reagent (Amersham) according to the manufacturer's instructions.

E. coli BL21(DE3) carrying desired pET28a-derived plasmids was cultured at 37°C in LB broth supplemented with kanamycin (30 μg/mL). When the OD₆₀₀ reached approximately 0.6 to 0.8, recombinant protein expression was induced by 0.2 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) and further incubated for 16 h at 18°C with constant shaking (220 rpm). Cells pelleted by centrifugation were resuspended in a lysis buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 10 mM imidazole, pH 8.0) and lysed via a high-pressure homogenization (JN-mini). After removing the cell debris by spinning at 20,000 × g for 30 min, the supernatants were incubated with 1 mL of prewashed Ni²⁺-nitrilotriacetic acid (NTA) beads (Qiagen) for 2 h at 4°C in a head-to-end rotator. Unbound proteins were cleared by the washing buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 8.0). Then, the His₆-tagged proteins were eluted by the elution buffer (50 mM NaH₂PO₄, 300 mM NaCl, and 250 mM imidazole, pH 8.0). The resulting proteins were dialyzed twice in a buffer containing 300 mM NaCl, 20 mM Tris-HCl (pH 7.5), and 10% glycerol. Protein concentrations were measured by the Bradford protein assay (Bio-Rad).

HK-2 cells were seeded at a density of 50,000–80,000 cells/well in specialized XF96 cell culture microplates (Seahorse Bioscience, Billerica, MA, United States). The cells were exposed to different conditions as required. Then, the medium was replaced with Seahorse running medium (XF base medium supplemented with 10% D-glucose, 100 mM pyruvate, and 200 mM glutamine for the mitochondrial stress test, or with 200 mM glutamine alone for the glycolysis stress test). Then, incubation was performed in a non-CO₂ incubator for 60 min at 37°C. The basal oxygen consumption rate and extracellular acidification rate were recorded for 24 min, followed by performance of the mitochondrial stress test (1 μM oligomycin, 2 μM carbonyl cyanide-p-trifluoromethox- yphenyl-hydrazon (FCCP), and 0.5 μM rotenone/antimycin A) and glycolysis stress test (10 mM glucose, 1 μM oligomycin, and 50 mM 2-Deoxy-d-glucose (2-DG)). Subsequently, the cells were lysed in RIPA buffer and subjected to the Bradford protein assay (Bio-Rad, Hercules, CA, United States). The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) values were normalized by the protein values in each well.

The Rho GTPase activation assay was performed using the G-LISA RhoA absorbance-based activation assay (Cytoskeleton^®). Briefly, cells were grown on collagen-coated 96-well plates (Costar^®), treated for 2 h with 20 µM of DMSO or 20 µM of MG-132 and incubated at 37 °C. At the end of the incubation period, all cells were washed twice with ice-cold PBS and re-suspended in 65 µl of G-LISA lysis buffer. Protein lysates were transferred to ice-cold 1.5-ml centrifuge tubes and clarified by centrifuging at 10,000 rpm for 2 min. Protein concentrations were determined using the Bradford Protein Assay (Bio-Rad^®), and 1.0 mg/ml protein was used for the Rho GTPase activation assay as per manufacturer’s recommendations. A 1:50 dilution of the primary antibody and 1:250 dilution of the HRP–conjugated secondary antibody were sufficient to produce a RhoA-specific signal. After antibody and HRP reagent incubation, signals were detected on a Versamax microplate reader at 490 nm (Molecular devices^®). Data analysis was performed using Microsoft Excel^®.