Bradford assay kit

The proteins were separated from each sample by Ready Prep TM protein extraction kit (Bio Rad Inc, USA), by RIPA lysis buffer (Bio BASIC INC, Canada) with additional protease inhibitor and phosphatase inhibitor buffer. Then, the samples were centrifuged at ∼16,000×g for 30 minutes at 4°C to separate the supernatant and the amount of the sample was measured with the Bradford Assay Kit (Bio Basic Inc, Canada). After the separation of the antigen samples by sodium dodecyl sulfate-polyacrylamide, gel electrophoresis (SDS-PAGE) gel (Bio-Rad Inc, USA) to the immunoblotting membrane (PVDF) membrane blocked with 3% bovine serum albumin (BSA) using Bio-Rad Trans-Blot Turbo. The MDM2 and E-cadherin proteins were incubated with their diluted primary at 4°C overnight and washed with TBST and secondary antibodies conjugated with HRP enzymes. Then, chemiluminescent signals were measured and the bands were analyzed using ChemiDoc MP Imager against beta-actin [23 (link)].

NAD⁺/NADH assay was performed as previously published^{2 (link)}, view detailed protocol in Supplemental materials and methods. ATP production capacity of isolated mitochondria was measured using an ATP determination kit (ThermoFisher Scientific) as per the manufacturer’s instructions (Supplemental Materials and Methods). ATP production for each sample was normalized to total protein using Bradford assay kit (BioBasic).

Proteins were extracted from kidney tissue samples by RIPA buffer containing a cocktail of protease inhibitors (Bio-Rad Inc.). Protein was quantified using a Bradford assay kit (SK3041; Bio Basic Inc., Markham Ontario L3R 8T4 Canada), then separated by 10% SDS-PAGE (Bio-Rad Laboratories Inc. Cat#161-0181), and transferred to PDVF membranes. Membranes were immunoblotted with indicated primary antibodies prepared at 5% blocking buffer (1 : 1000) and were cultured overnight at 4°C. After extensive washing in TBS buffer, the membranes were incubated with the corresponding HRP-conjugated secondary antibodies (Goat anti-rabbit IgG-HRP-1 mg Goat mab-Novus Biologicals) and developed using the chemiluminescent substrate (ClarityTM Western ECL substrate Bio-Rad Cat#170-5060). β-Actin was used as a loading control. Image analysis software was assessed by normalization procedure of each phosphorylated active target protein versus corresponding control sample total protein on the ChemiDoc MP imager.
Primary antibodies contained antibody t-PI3K (Cat#3358), p-PI3K (sc-1637), t-Akt (Cat#9272), p-Akt (Cat#9271), t-p65 (Cat#3034), p-p65 (Cat#3033,), t-NF-κB inhibitor α (IκBα, Cat#9242), p-IκBα (Cat#2859), cleaved-caspase-3 (Cat#AB3623), and β-actin (sc-8432).