Ab62352

For the NRF2 ubiquitination determination, the cells were harvested with RIPA buffer (9806, Cell Signaling, Danvers, MA, USA) and the lysates were submitted to three freeze–thaw cycles. After centrifugation, the supernatants were collected for use and the protein concentration was determined with Pierce BCA Protein Assay Kit (23225, Thermo Fisher Scientific, Waltham, MA, USA). The NRF2 was immunoprecipitated from the total cell lysates (containing 1 mg of total protein) with 8 μg of anti-NRF2 antibody (PM069, MBL International Corporation, Woburn, MA, USA) and the NRF2 ubiquitination was analyzed by Western blot with anti-ubiquitin antibody (AUB01-HRP, Cytoskeleton, Denver, CO, USA). The membranes were stripped and reprobed with anti-NRF2 antibody (ab62352, Abcam, Cambridge, UK) to determine the levels of immunoprecipitated NRF2. A sample, immunoprecipitated with 8 μg of Normal Rabbit IgG (2729, Cell Signaling, Danvers, MA, USA), was used as a control for nonspecific bindings. A sample (40 μg of protein) of the pre-immunoprecipitation lysate was also analyzed by Western blot with anti-NRF2 antibody (ab62352, Abcam, Cambridge, UK) to show levels of NRF2 before immunoprecipitation (input). The densitometric analysis was completed using the UVP VisionWorks LS Analysis Software (UVP, Upland, CA, USA). The ubiquitinated NRF2 was normalized by immunoprecipitated NRF2.

The clinical tissue samples used in this study were histopathologically and clinically diagnosed at Fudan University Shanghai Cancer Center. Prior patient consent and approval from the Institutional Research Ethics Committee were obtained. Paraffin‐embedded tissue slides were deparaffinized in xylene, rehydrated through graded alcohol solutions, blocked in methanol containing 3% hydrogen peroxide, and then incubated with dCK and NRF2 antibodies. The dCK antibody (Abcam, ab96599) was used at a dilution factor of 1:50. The NRF2 antibody (Abcam, ab62352) was diluted to a ratio of 1:100, and then, the slides were rinsed in PBS solution and incubated with secondary antibodies and peroxidase reagent at room temperature. Finally, the slides were incubated with 3,3′‐diaminobenzidine solution at room temperature for 10 minutes and counterstained with haematoxylin. A scoring scale was used to evaluate the percentage of stained cells (0, <10%; 1, 10%‐25%; 2, 25%‐50%; 3, 50%‐75%; 4, >75%) and the staining intensity (0, negative; 1, low; 2, moderate; 3, strong). The overall staining scores were determined by combining the two scores (frequency × intensity). An immunohistochemical score >6 was defined as high expression, whereas a score ≤6 was considered a low expression level.

Slides were then incubated with specific primary antibodies against a-SMA (rat, Ab5694, 1 : 200, Abcam, USA) and Nrf2 (Ab62352, 1 : 200, Abcam, USA) at 4°C overnight. Next day, after washing with 1x PBS for 5 min/3times, slides were incubated with secondary antibody (1 : 500, Boster, China) for 1 h at room temperature. Then, samples were stained with DAPI (AR1177, BOSTER, China) and sealed in antifade fluorescence mounting medium (AR1109, BOSTER, China) with coverslips. The detection of a-SMA and Nrf2 proteins was performed under a fluorescence microscope (IX71, Olympus, Japan). Nrf-2 positive cells were counted using Image pro plus software. The percentage of Nrf-2 in the nucleus was quantitatively analyzed by the ratio of nuclear Nrf-2 positive cells to total cells in each field.