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.
Visualizing HaloTagged AP-2 in Eps15 Knockout Cells
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.
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Corresponding Organization : The University of Texas at Austin
Other organizations : The University of Texas Health Science Center at San Antonio
Variable analysis
- Gene editing to add a HaloTag to both alleles of AP-2 σ2
- Gene editing to knock out both alleles of endogenous Eps15 using CRISPR-associated protein 9 (Cas9)
- Visualization of HaloTagged AP-2 σ2
- Cell culture media composition (1:1 DMEM high glucose: Ham's F-12, 5% fetal bovine serum, Penicillin/Streptomycin/l-glutamine, 1 μg ml^-1 hydrocortisone, 5 μg ml^-1 insulin, 10 mM HEPES, pH 7.4)
- Cell seeding density (3 × 10^4 cells per coverslip)
- Transfection reagent (3 μl Fugene HD)
- Transfected plasmid DNA (1 μg)
- Imaging conditions (37 °C, 5% CO2)
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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