Chemidoc mp video documentation system

The protein level of heat shock proteins (HSP60 and HSP70) and minichromosome maintenance complex 2 (MCM2) was measured after 24 and 72 h of HeLa cells incubation with 50 µg/mL derivatives. Samples corresponding to 100 µg of protein were separated on a 10% SDS/PAGE under reducing conditions and transferred to 0.4 µm (Millipore) polyvinylidene difluoride (PVDF) membrane in a wet transfer system (Bio-Rad, Hercules, CA, USA). Membranes were blocked with the blocking solution included in the Western Breeze Chromogenic kit (Invitrogen) for 30 min at room temperature. Detection was performed with mouse monoclonal anti-HSP60, anti-HSP70, and anti-MCM2 primary antibodies (1:250 dilution Santa Cruz Biotechnology). Further, the membranes were processed according to the manufacturer’s instructions, using alkaline phosphatase-conjugated anti-mouse and anti-rabbit secondary antibodies and 5-bromo-4-chloro-3′-indole phosphate/nitroblue tetrazolium as a chromogenic substrate (Invitrogen by Thermo Fisher Scientific). The resulting bands were visualized and photographed using a transilluminator (ChemiDoc MP Video Documentation System, Bio-Rad) and were densitometered using the GelQuant.NET software version 1.7.8.

The protein level of p53, Beclin-1, LC-3, and Nrf-2 was determined in the samples collected from the control and 100 mg of SiQD/kg of b.w. after 1, 6, 24, and 72 h. Cell lysates corresponding to 100 µg of protein were separated on a 10% SDS/PAGE under reducing conditions and transferred to 0.4 µm polyvinylidene difluoride (PVDF) membrane in a wet transfer tank (Bio-Rad, Hercules, CA, USA). Membranes were blocked with the blocking solution included in the WesternBreeze Chromogenic kit (Invitrogen, Rockford, IL, USA) for 30 min at room temperature. The detection of p53, LC-3, and Nrf-2 proteins was performed with the rabbit polyclonal anti-p53, anti-LC-3, and anti-Nrf-2 primary antibodies, respectively (1:250 dilution). The highlight of Beclin-1 protein was performed using anti-Beclin-1 mouse polyclonal primary antibody (1:250 dilution, SantaCruz Biotechnology, Dallas, TX, USA). After the incubation with primary antibodies, membranes were processed according to the manufacturer’s instructions, using alkaline phosphatase-conjugated anti-mouse and anti-rabbit secondary antibodies and 5-bromo-4-chloro-3′-indolephosphate/nitroblue tetrazolium as the chromogenic substrate. The resulting bands were visualized and photographed using a transilluminator (ChemiDoc MP Video Documentation System, Bio-Rad, Hercules, CA, USA) and were analyzed using the GelQuant.NET software version 1.7.8.