Human-derived SUM159 cells gene-edited to add a HaloTag to both alleles of AP-2 σ2 were a gift from T. Kirchhausen54 (link). Cells were further gene-edited to knock out both alleles of endogenous Eps15 using CRISPR-associated protein 9 (Cas9) to produce the Eps15 knockout cells developed previously by our group12 (link).
Cells were grown in 1:1 DMEM high glucose: Ham’s F-12 (Hyclone, GE Healthcare) supplemented with 5% fetal bovine serum (Hyclone), Penicillin/Streptomycin/l-glutamine (Hyclone), 1 μg ml−1 hydrocortisone (H4001; Sigma-Aldrich), 5 μg ml−1 insulin (I6634; Sigma-Aldrich) and 10 mM HEPES, pH 7.4 and incubated at 37 °C with 5% CO2. Cells were seeded onto acid-washed coverslips at a density of 3 × 104 cells per coverslip for 24 h before transfection with 1μg of plasmid DNA using 3 μl Fugene HD transfection reagent (Promega). HaloTagged AP-2 σ2 was visualized by adding Janelia Fluor 646- HaloTag ligand (Promega). Ligand (100 nM) was added to cells and incubated at 37 °C for 15 min. Cells were washed with fresh medium and imaged immediately.
Cells were grown in 1:1 DMEM high glucose: Ham’s F-12 (Hyclone, GE Healthcare) supplemented with 5% fetal bovine serum (Hyclone), Penicillin/Streptomycin/l-glutamine (Hyclone), 1 μg ml−1 hydrocortisone (H4001; Sigma-Aldrich), 5 μg ml−1 insulin (I6634; Sigma-Aldrich) and 10 mM HEPES, pH 7.4 and incubated at 37 °C with 5% CO2. Cells were seeded onto acid-washed coverslips at a density of 3 × 104 cells per coverslip for 24 h before transfection with 1μg of plasmid DNA using 3 μl Fugene HD transfection reagent (Promega). HaloTagged AP-2 σ2 was visualized by adding Janelia Fluor 646- HaloTag ligand (Promega). Ligand (100 nM) was added to cells and incubated at 37 °C for 15 min. Cells were washed with fresh medium and imaged immediately.
