Cell lysates of ALDH−, ALDH+, and CD44+/CD24− cells following treatment with vehicle and treatment (ASR490, DAPT, MG132, CHX, or CQ) for prescribed doses and time points, were prepared with RIPA buffer (Thermo Scientific, Rockford, IL, United States) per the manufacturer’s protocol. Western blotting was performed using specific antibodies against Notch1 (CST, #3608), HES1 (Sigma, #SAB2108472), Hey1 (Proteintech, #19929-1-AP), NFκB p65 (CST, #8242), Bcl-2 (CST, #15071), Bcl-xL (CST, #2764), Vimentin (CST, #46173), Slug (CST, #9585), E-Cadherin (CST, #3195), β-catenin (CST, #8480), Ubiquitin (CST, #3933), Cleaved-PARP (CST, #5625), Cleaved-caspase-9 (CST, #20750), BAX (CST, #41162), Notch2 (CST, #D76A6), Lamp1 (CST, #9091), and LC3B (Proteintech, #14600-1-AP). β-Actin (CST, #4970) was used as the loading control. Protein bands were visualized using the Bio-Rad ChemiDocTM imaging system. For IP experiments, protein samples were immunoprecipitated with Notch1 antibody as per the protocol described elsewhere (Chandrasekaran et al., 2020 (link)), and Western blots were performed with ubiquitin antibody.
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