Chatterjee et al. AhR Western blot Cell Reports 2023

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These figures depict western blot results for AhR and b actin protein visualisation and quantification upon CRISPR/Cas9 gene editing and/or TCR triggering: Figure 1B, Figure 1C, Supplemental Figure 1A and Supplemental Figure 2C.

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Western Blot Method Cells were lysed with the RIPA buffer (Cell Signaling) supplemented with phosphatase inhibitors (PhosSTOP) and protease inhibitor cocktail (Complete Mini EDTA-free, Roche) for 10 minutes at 4 °C and centrifuged at 14,000 g for 10 minutes. Proteins were quantified by Bradford assay (Bio-Rad, Hercules, CA, USA). Samples (15-30 µg protein/well) were loaded on SDS PAGE gel (90 minutes, 100 V), transferred to PVDF membrane (Millipore) and blocked with 5% BSA. Subsequently membranes were incubated with primary AhR antibodies (Clone ab190797, Abcam, Cambridge, UK) and β-actin (Sigma, USA). Immunoreactive bands were detected using HRP-conjugated secondary antibodies and revealed with ECL substrate (GE Healthcare, Chicago, IL, USA) (Supplemental Tables 2; Key Resources Table). See manuscript by Chatterjee D et al., accepted in Cell Reports, 2023.

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