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.
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.
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