Enhanced chemiluminescence reaction kit

We carried out the Western blot analysis as described previously (28 (link)). In brief, cells were treated with 0.6 μM of erdafitinib for 0, 24, 48, or 72 h at 37°C, and lysed. Equal amounts (10 μg) of proteins were subjected to SDS-PAGE and then transferred to PVDF membranes. The membranes were incubated with anti-ABCB1 (1:1000) and anti-GAPDH (1:1000) overnight at 4°C. After incubating with secondary antibody (1:2000), the bands were analyzed using the enhanced chemiluminescence reaction kit (Amersham, NJ).
Immunofluorescent staining was performed as reported previously (18 (link)). In brief, cells were seeded in 24-well plate, and then treated with 0.6 μM of erdafitinib for 0, 24, 48, or 72 h. Briefly, the cells were fixed using 4% formaldehyde for 15 min and then incubated with ABCB1 antibody (1:500) overnight. Subsequently, cells were incubated with Alexa Fluor 488 conjugated IgG secondary antibody (1:2000) and DAPI. The cells were visualized using a Nikon TE-2000S fluorescence microscope (Nikon Instruments Inc., Melville, NY) and photographs were taken.

Following protein isolation, equivalent amounts of protein were loaded onto a 10% (for inducible nitric oxide synthase (iNOS)) and 12% (for caspase-3) polyacrylamide gel for electrophoresis and blotting. After being blocked for 1 h at ambient temperature, the membrane was incubated overnight at 4°C with polyclonal anti-mouse iNOS antibody (BD Transduction Laboratories, Lexington, KY, USA) or polyclonal anti-rabbit cleaved caspase 3 antibody (Cell Signaling Technology, Danvers, CO, USA) at a 1: 1000 dilution. The blots were washed and incubated with horseradish-peroxidase-coupled secondary antibody (diluted 1∶1500; BD Transduction Laboratories, Lexington, KY, USA) for 1 h at ambient temperature. The blots were then stripped and reprobed with β-actin antibody (1∶3000; BD Transduction Laboratories, Lexington, KY, USA) as internal control. Immunoreactivity was visualized using an enhanced chemiluminescence reaction kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) and quantified by scanning densitometer.

A bicinchoninic acid protein assay kit (Thermo Fisher Scientific) was utilized to measure the protein concentrations in whole cell lysates. Next, SDS‐PAGE (20 µg per lane) was used to separate the proteins before blotting them onto 0.2 µm PVDF membranes. The membranes were subjected to incubation with primary antibodies for one night after being blocked with 5% nonfat milk. The next step was to incubate the membranes for 1 h with secondary antibodies that were conjugated to HRP. An enhanced chemiluminescence reaction kit (Amersham Biosciences) was used to detect protein bands.