bsa anti nrf2  (Cell Signaling Technology Inc)

Journal: Redox Biology

Article Title: Development of KEAP1-targeting PROTAC and its antioxidant properties: In vitro and in vivo

doi: 10.1016/j.redox.2023.102783

Figure Lengend Snippet: SD2267 promoted the nuclear translocation of NRF2 and activated NRF2 target genes in AML12 cells (A) AML12 cells were incubated with 300 nM of SD2267 for the indicated times, and subjected to nuclear fractionation. GAPDH was used as a loading control for cytosolic proteins, and Lamin B was used as a loading control for nuclear proteins. Protein expression levels were determined by western blotting, and densitometry was performed using three independent experiments. ** p < 0.01, *** p < 0.001. (B) AML12 cells were incubated with 100 or 300 nM of SD2267 for 3 h, and subjected to NRF2 immunofluorescence staining (green). DAPI was used to stain nuclei (blue). In merged NRF2 and DAPI images, nuclear NRF2 stained blue-green (Scale bar = 25 μm). (C) AML12 cells were incubated with 100 nM of SD2267 for the indicated times. Gene expression levels of HMOX1 , NQO1 , GCLC , and GCLM were measured by qRT-PCR. Relative expression levels are presented as mean ± SEM (n = 3 per group). ** p < 0.01, *** p < 0.001, ns; not significant versus the control. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: After rinsing with PBS-T (0.1% (v/v) Tween 20 in PBS), cells were blocked with 1% (w/v in PBS-T) BSA for 1 h, incubated with anti-NRF2 antibody (Proteintech, 16396-1-AP, IL, USA) (1:200 in 1% BSA) overnight at 4 °C, washed with PBS-T, and incubated with donkey anti-rabbit IgG FITC (Santa Cruz Biotechnology, SC-2090, 1:200 in 1% BSA) for 1 h at room temperature.

Techniques: Translocation Assay, Incubation, Fractionation, Control, Expressing, Western Blot, Immunofluorescence, Staining, Gene Expression, Quantitative RT-PCR