Image station 4000r pro

H661 cells, plated at 6,000 cells/cm²/3 mL in 6-well plates, were incubated for 24 hours at 37° C in 5% CO₂ humidified atmosphere, synchronized, pretreated with lunasin, and released as described for cell cycle analysis. At 2 to 3 hour intervals over a 30 hour period, cells were harvested and prepared for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis using standard methods. Briefly, cells were harvested by scraping, lysed in RIPA buffer, the protein concentration determined using the Pierce™ BCA Protein Assay Kit (Thermo Scientific) with BSA as a standard, and the total protein adjusted to 2 mg/mL in reducing sample loading buffer. Samples (7.5 μg protein/well) were subjected to SDS-PAGE, transferred by electroblotting to polyvinylidene difluoride membrane and probed with the relevant antibodies. Luminescent detection utilized the chemiluminescent substrate SuperSignal® West Femto while colorimetric detection utilized 1-Step™ NBT/BCIP (nitro-blue tetrazolium/5-bromo-4-chloro-3′-indolyphosphate) both from Thermo Scientific. Immunoblots were imaged using a Kodak Image Station 4000R Pro utilizing Carestream Molecular Imaging Software v5.0.7.24 (Carestream). Densitometry was performed using the ImageJ software v1.45.

SDS-PAGE was used to separate and analyze the proteins after extraction. To 45 μL of each sample, 15 μL of 4X SDS loading dye with 2-mercaptoethanol was added. A gel was loaded with the samples and a ladder in 1X XT MES buffer. Electrophoresis was at 200 V for 45 min. Coomasie stain was poured onto the gel, microwaved for 30 s, and rotated at 60 rpm for 25–30 min. The gel was then rinsed in DI water, and the destain was added to the gel. The gel was microwaved for 30 s and rotated at 60 rpm for 30 more minutes. Gels were imaged on a Kodak Image station 4000R Pro and densitometry analysis was done using Carestream SE M software to analyze amount and size of the proteins found in the samples. A standard curve was generated using known concentrations of GRFT (1, 3, 5, 10 μg) vs. their gel intensity. Unknown samples were then fit to the curve and concentrations were calculated.