Full length caspase 3

For western blotting analysis, 40 μg protein per sample was loaded on a 10% gel and resolved using SDS-PAGE. The proteins were transferred to a PVDF membrane; the membranes were blocked using 5% milk and subsequently incubated with rabbit anti-NOX2, NOX4, full-length caspase-3, cleaved caspase-3, and β-actin antibodies (Santa Cruz Biotechnology, Inc.) at 4°C overnight. Subsequently, the membrane was washed and incubated with secondary antibodies (horseradish peroxidase-conjugated) at 4°C, and the protein bands were visualized using an enhanced chemiluminescence kit (Amersham Biosciences). Finally, the expression levels of protein were calculated using a Molecular Imager ChemiDoc XRS system (Bio-Rad Laboratories, Inc.). β-Actin was used as the loading control.

Cells (1 × 10⁶ cells in 10 ml medium) were seeded in 10-cm dishes. After the NSCLC cells were treated with DMSO or TBPT, they were washed and collected. Cell lysates were prepared conventionally in RIPA lysis buffer. The protein concentrations were determined using the BCA protein assay. Equal amounts of total protein were loaded onto SDS-PAGE gels (8–10%) and transferred onto PVDF membranes. After the membranes were blocked with 5% nonfat milk for 1 h, they were blotted with primary antibodies against P-gp, β-actin, full-length caspase 3 (Santa Cruz Biotechnology, Dallas, TX, USA), caspase 8 (BD Biosciences, San Jose, CA, USA), p21, caspase 9, cleaved caspase 3 and PARP (CST) at 1 : 500–1 : 1000 dilution overnight at 4 °C. Then, the membranes were incubated with goat anti-mouse or goat anti-rabbit secondary antibodies (1 : 5000, Santa Cruz) at RT for 1 h. An enhanced chemiluminescence western blot system (Millipore) was used to detect the immunoreactive bands. Intensity of the blot was determined using the ImageJ software (NIH, Bethesda, MD, USA).