Human-derived SUM159 cells gene-edited to add HaloTag (Promega) to both alleles of AP-2 σ2 were a generous gift from Tom Kirchhausen29 (link). Cells were grown in 1:1 DMEM high glucose:Ham’s F-12 (Hyclone, GE Healthcare) supplemented with 5% fetal bovine serum (Hyclone), Pen/Strep/L-glutamine (Hyclone), 1 μg/ml hydrocortisone (H4001; Sigma-Aldrich), 5 μg/ml insulin (I6634; Sigma-Aldrich), and 10 mM HEPES (Fisher), pH 7.4 and incubated at 37°C with 5% CO2. Cells were seeded onto acid-washed coverslips at a density of 3x104 cells per coverslip for 24 h before transfection with 1-3 μg of plasmid DNA using 3 μL Fugene HD transfection reagent (Promega).
HaloTagged AP-2 σ2 was visualized by adding Janelia Fluor 646-HaloTag ligand, a gift from Luke Lavis30 . 100 nM ligand was added to cells and incubated at 37°C for 15 minutes. Cells were washed with fresh media and imaged immediately.
HaloTagged AP-2 σ2 was visualized by adding Janelia Fluor 646-HaloTag ligand, a gift from Luke Lavis30 . 100 nM ligand was added to cells and incubated at 37°C for 15 minutes. Cells were washed with fresh media and imaged immediately.
