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. 2025 Aug 6;86:103815. doi: 10.1016/j.redox.2025.103815

Fig. 4.

Fig. 4

CTSS knockdown facilitates degradation of NRF2 mediated by the K48-linked ubiquitination. (A) The half-life of NRF2 protein expression in CTSS knockdown cells and parental control cells was measured by western blot assay. HCC cells were treated with 20 μg/mL cycloheximide (CHX) for the indicated time before collection. (B) Western blot analysis of NRF2 protein expression in the indicated cells untreated or treated with MG-132. For MG-132 treatment, PLC/PRF/5 and Hep3B cells were pre-treated with 20 μM MG-132 for 8 h before collection. (C) The endogenous ubiquitination levels of NRF2 in the indicated cells were measured by western blot assay. HCC cells were pre-treated with 20 μM MG-132 for 8 h before being lysed and anti-NRF2 primary antibodies were added, followed by immunoblotting with anti-ubiquitin antibody. (D) The exogenous ubiquitination levels of NRF2 in HEK-293T cells were measured by western blot assay. HEK-293T cells were co-transfected with the indicated plasmids. After 48 h of transfection, 20 μM MG-132 was added for 8 h before being lysed and immunoprecipitated with anti-MYC primary antibodies, followed by immunoblotting with anti-HA antibody. (E–F) The endogenous K48-linked and K63-linked ubiquitination levels of NRF2 in the indicated cells were measured by western blot assay. HCC cells were pre-treated with 20 μM MG-132 for 8 h before being lysed, and anti-NRF2 primary antibodies were added, followed by immunoblotting with anti-K48-ubiquitin and K63-ubiquitin antibodies. (G) The exogenous K48-linked and K63-linked ubiquitination levels of NRF2 in HEK-293T cells were measured by western blot assay. HEK-293T cells were co-transfected with the indicated plasmids (HA-tagged K48-Ub and K64-Ub plasmids referred to other lysines were mutated into arginin except K48 or K63 residue). After 48 h of transfection, 20 μM MG-132 was added for 8 h before being lysed and immunoprecipitated with anti-MYC primary antibodies, followed by immunoblotting with anti-HA antibody. All data are acquired from at least three independent experiments and presented as the mean ± SD. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.