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Immunoblotting and electrophoretic mobility shift assay After each treatment, cell proteins were fractionated by SDS gel electrophoresis and immunoblotted with antibodies against Trx-1 (AbCam, Cambridge, MA, USA), B-cell lymphoma 2 (Bcl-2) and myeloid cell leukemia 1 (Mcl-1) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and ASK1, phospho-p38, phospho-extracellular signal-regulated kinase (phospho-ERK) and phospho-Jun N-terminal kinase (phospho-JNK) (Cell Signaling, Danvers, MA, USA) following standard procedures

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Additionally, genetic and epigenetic variability among patients, resistance mechanisms, potential side effects, and the difficulties of translating preclinical findings to clinical applications further complicate the development of effective NRF2-targeted therapies