M35 a x omat processor

Western blotting was performed as described previously.45, 46 Briefly, cells were collected and lysed with RIPA buffer (Thermo Scientific, MA, USA). Approximately, 20 μg protein was loaded and run on sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). The proteins were transferred onto polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% skim milk and subsequently incubated with the specific primary antibodies overnight at 4°C with gentle shaking. The membrane was then probed with ECL system (AbClon) for signal detection. Films were developed using a Kodak M35‐A X‐OMAT processor.

Antibodies used were LONP1 antibody (15440-1-AP, ProteinTech Group, Inc.), HIF-1α (GTX127309, GeneTex), COX IV (ab62164, Abcam) and β-actin (NB600-501, Novus). The images were exposed by KODAK M35A X-OMAT Processor.

Cells (3 × 10⁵) were seeded in a six-well plate and treated with respective formulations for 24 h. After treatment, the cells were harvested and lysed on ice for 30 min. The whole cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoresed samples were blotted onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% skim milk and then incubated with respective primary antibody overnight at 4 °C. Subsequently, the membranes were washed in TBST buffer and incubated with secondary antibody conjugated to horseradish peroxidase for 2 h. The membranes were then developed for signal detection and imaged using Kodak M35-A X-OMAT processor.

Cells were harvested and protein lysate was collected as previously described [46 (link)]. The protein concentration was determined by Bicinchoninic acid assay kit (Thermo Scientific, Rochester, USA) and 25 μg proteins were separated through SDS-PAGE using 4–12% Bis-Tris gel. The separated proteins were transfered onto PVDF membrane (GenHunter Corp, Nashville, USA). The membrane was blocked with 5% non-fat milk and probed with UBE2N/Ubc13 primary Ab (1:1000, R&D Systems, UK). The membrane was incubated with secondary Ab conjugated with HRP, incubated with ECL western blotting substrate and exposed on CL-Xposure films (Thermo Scientific, Rochester, New York). Films were revealed using a Kodak M35-A X-OMAT processor.

Proteins were extracted and fractionated by SDS-PAGE as previously described 7. Briefly, nuclear and cytoplasmic fractions were extracted using a NE-PER kit per manufacturer recommendations (Pierce Rockford, IL), protein contents estimated using BCA assay (Pierce). Western blot assays were performed using 15 µg of protein samples, resolved on 4–12% NU-PAGE Bis-Tris acrylamide gel (Invitrogen) and proteins transferred on a PVDF membrane (GenHunter, Nashville, TN) and subjected to Suz12 (Millipore, cat#17661, 1:1000 dilution) or PCNA (AbCam cat#18197, 1:1000 dilution) detection. We used secondary antibody goat anti-mouse or rabbit conjugated with HRP (Abcam, cat# ab6789 and ab6721 respectively) diluted 1:10,000. Once incubated with enhanced chemiluminescent substrate (Pierce), CL-Xposure films (Thermo Scientific) were exposed and revealed using a Kodak M35-A X-OMAT processor. Band intensity was evaluated by ImageJ software.