HEK 293T, HeLa, and adherent HeLa S3 cells were cultivated under standard conditions (37°C and 5% CO2 atmosphere) by using Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 10% FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). Adapted HeLa S3 cells were grown in Joklik's medium (Sigma-Aldrich) supplemented with l -glutamine, nonessential amino acids, sodium hydrogen carbonate, 10% FBS and 1% penicillin–streptomycin in a rotating cell culture system at 37°C. Sf21 cells were cultured in SF-900 II SFM serum-free medium (Gibco) at 27°C, shaking at 80 r.p.m.
For immunoprecipitations of overexpressed proteins, HEK 293T cells were cultured on dishes with a diameter of 15 cm. Then cells were calcium phosphate-transfected with 4–10 µg of plasmid DNA per plate. Cells were harvested 48 h after transfection. For immunofluorescence, HEK 293T and HeLa cells were transfected with 125–500 ng of plasmid DNA at a confluency of 100% on 24-wells using Lipofectamine 2000 (Thermo Fisher Scientific). Twenty four hours after transfection, 50% of transfected HeLa cells were transferred on cover slips. For baculoviruses amplification, Sf21 cells were seeded on 6-wells at a cell count of 0.5 × 106 cell/mL and transfected with FuGENE (Promega).
For immunoprecipitations of overexpressed proteins, HEK 293T cells were cultured on dishes with a diameter of 15 cm. Then cells were calcium phosphate-transfected with 4–10 µg of plasmid DNA per plate. Cells were harvested 48 h after transfection. For immunofluorescence, HEK 293T and HeLa cells were transfected with 125–500 ng of plasmid DNA at a confluency of 100% on 24-wells using Lipofectamine 2000 (Thermo Fisher Scientific). Twenty four hours after transfection, 50% of transfected HeLa cells were transferred on cover slips. For baculoviruses amplification, Sf21 cells were seeded on 6-wells at a cell count of 0.5 × 106 cell/mL and transfected with FuGENE (Promega).