Luminata crescendo horseradish peroxidase substrate

The cells were treated similarly as in the Annexin V-PI apoptosis analysis. After 24 h of treatment, cells were lysed in radioimmunoprecipitation buffer with protease inhibitor mixture (5871, Cell Signaling Technology) by incubating for 30 min on ice. After centrifugation (14,000 × g for 15 min at 4 °C), the protein amount in the supernatant was quantified using BCA Protein Assay kit (23225, Thermo Fisher). Then, the protein was diluted with 4× LDS sample buffer (NP0007, Thermo Fisher), electrophoresed on a NuPage Novex 4–12% Bis-Tris Gel (NP0335BOX, Thermo Fisher), and transferred onto polyvinylidene difluoride membrane (29301-854, VWR International). Primary and secondary antibodies used for immunoblotting are listed in SI Appendix, Table S6. Incubation with primary antibodies (AHR, SLC7A5) was performed at 4 °C in 5% nonfat milk (170-6404, Bio-Rad) overnight. Anti–β-actin (ACTB) was used as a loading control. The membranes were then washed and incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Detection was performed by using Luminata Crescendo horseradish peroxidase substrate (WBLUR0500, Millipore) or SuperSignal West Femto Substrate (34096, Thermo Fisher). Immunoblot images were captured by iBright CL1500 imaging system (Thermo Fisher) and each protein was quantified using ImageJ software.