Small-molecule modulation of β-arrestins

Cell culture, antibodies, and reagents HEK-293 cells (ATCC), including transient and stable lines, as well as CRISPR–Cas9 βARR1/2-knockout and parental lines 61 , were maintained in Eagle’s Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C and 5% CO 2 . U2OS cells (DiscoveRx PathHunter) were cultured in MEM containing 2 mM l -glutamine, 10% FBS, and 1% penicillin-streptomycin under similar conditions. U2OS-based β-arrestin recruitment and internalization assays were performed per the manufacturer’s protocol (DiscoveRx). For chemokine-induced migration assays, leukocytes were isolated from wild-type mice as described previously 18 , 37 , 61 . Escherichia coli strains DH5α and BL21 (DE3) (New England Biolabs) were cultured in LB or Terrific Broth (Fisher Scientific) at 37 °C. DH5α was used for plasmid amplification, and BL21 was used for recombinant protein expression. Sf9 insect cells ( Spodoptera frugiperda , Expression Systems, 94-001F) were cultured in ESF 921 medium (Expression Systems) at 27 °C. Baculoviruses were generated and amplified according to the manufacturer’s instructions. For serum starvation experiments, cells were cultured in serum-free medium supplemented with 0.1% BSA, 10 mM HEPES, and 1% penicillin-streptomycin. Transfections were performed using FuGene 6 (Promega) or Lipofectamine 3000 (Invitrogen), according to manufacturers’ protocols. …