Cocktail of proteinase inhibitors

The control samples consisted with MCF-7 and A549 cells treated with different doses of RSV or DMSO. Total cell proteins were prepared at 24 h post treatment using cell lysis buffer (Cell Signaling Technology, Danvers, USA) supplemented with a cocktail of proteinase inhibitors (Sigma-Adrich). The Western blot analysis was performed as described in a previously published study by our research group [21 (link)]. Briefly, total protein was resolved by SDS-PAGE and transferred onto a polyvinylidene difluoride membranes (Millipore, USA) and blocked with 5% nonfat dry milk. The membrane was incubated with a primary antibody, followed by a secondary antibody coupled to HRP. The target bands were developed with ECL substrate (WBKLS0500; Millipore, USA). All experiments were repeated at least three times. The band intensities in the immunoblotting were determined by ImageJ software.

Cellular extracts were prepared after 4 h of treatment with DSBA and fractionation of cytosolic and nuclear proteins that was carried out with a Thermo Scientific NE-PER Nuclear and Cytoplasmic Extraction Kit (Cat# 78833, Thermo Fisher). Protein samples were extracted using cell lysis buffer (Cell Signaling) supplemented with a cocktail of proteinase inhibitors (Sigma) and protein concentrations were determined with the Bio-Rad Dc protein assay kit (Bio-Rad Laboratories). Western blots was performed as described in [16 (link)]. Briefly, 50 μg of protein samples were resolved on 10% Mini-Protean TGX gels (Bio-Rad) and transferred onto 0.2 mM PVDF membrane (Millipore). Blots were blocked with 5% nonfat milk for 1–2 h at room temperature, then probed with primary antibodies, and incubated at 4°C overnight. Primary antibodies used were: anti-GSTP (#3369), anti-Nrf2 (#12721) and anti-aldehyde dehydrogenase-1 (ALDH1) (#12035) from Cell Signaling; heme-oxygenase 1 (HO-1) (SC-390991), anti-Nrf2 (SC-772) and Tubulin (SC-23948) from Santa Cruz Biotechnology. After extensive washing with TBST, blots were incubated with appropriate HRP-conjugated secondary antibody for 1.5 h at room temperature. Protein bands were detected using an ECL Plus Western Blot Detection System (GE Healthcare Life Science).

Western blot analyses were performed by lysing SK-MEL-28 and A375 cells with Laemmli buffer supplemented with a cocktail of proteinase inhibitors (Sigma Aldrich, Saint Louis, MO, USA). After sonication, the quantification of the total protein amounts was determined by a Micro BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Cleveland, OH, USA). Proteins (30 μg protein/lane) were resolved by SDS-PAGE, transferred to 0.2 µm nitrocellulose membranes (BIO-RAD, Hercules, CA, USA), incubated for 1 h with 5% milk to block nonspecific protein sites, and then subjected to immunoblot assay. We then evaluated the expression of the proteins of interest: SMO (Santa Cruz, Dallas, TX, USA); GLI1; CAXII; and β-actin (Cell Signaling, Denver, CO, USA). Membranes were incubated with a horseradish peroxidase-conjugated appropriate secondary antibody (Cell Signaling); then, the antigen–antibody complexes were visualized using an Immunostar HRP kit (Bio-Rad Laboratories, Hercules, CA, USA). β-actin was used as housekeeping protein for protein expression. The chemiluminescence of the immunoreactive bands was detected with a CCD camera gel documentation system (ChemiDoc XRS apparatus, Bio-Rad Laboratories) and quantified with Image Lab software (Bio-Rad Laboratories). For original blots, see Supplementary File S1.