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Expression and purification of β2AR in Sf9 cells The β2AR construct PN1 was expressed and purified as previously described39,42. In brief, Sf9 cells were infected with a PN1-containing baculovirus produced using the BestBac method. Cells were then collected and resuspended in chilled lysis buffer containing 10 mM HEPES, pH 7.4, 1 mM EDTA, 1 μM alprenolol and protease inhibitors
RNA sequencing and analysis E. coli BL21(DE3) cells (NEB: C2527H), which lack endogenous copies of IS621 elements, were transformed with plasmids encoding either the RE–LE junction or the LE–recombinase–RE region of the IS621 element. The cells were plated on LB (lysogeny broth) agar with kanamycin and grown overnight at 37 °C. Colonies were scraped from the
Compounds dBET6 (HY-112588), dBRD9 (HY-117690), MLN7243 (HY-100487), MLN4924 (HY-70062), MG132 (HY-13259), bafilomycin A1 (HY-100558), and 5K Scaffold Library (HY-L902) were obtained from MedChemExpress. Plasmids The following plasmids were used in this study: Artichoke (Addgene #73320) and Cilantro (Addgene #74450) for flow-based reporter degradation assays, reporter CRISPR screens and co-immunoprecipitation; sgBFP (U6.sgRNA.SFFV.tBFP) for validation of DCAF11-knockout
