HEK 293T and HEK TRex cells were from Invitrogen and cultured in DMEM (Gibco) containing 10% Fetal Calf Serum (FCS). The cells were routinely tested for mycoplasma and found to be negative. Cells were not authenticated. For the analysis of stalling on polybasic sequences, cells were first plated on 24-well plate and transfected 24 h later with dual-fluorescent reporter constructs using TransIt 293 reagent (Mirus) according to manufacturer’s protocol. After 20 h of expression, cells were washed with PBS and trypsinised. After inhibiting trypsin, cells were collected into 1.5 ml tubes, spun at 5,000 rpm for 3 min at 4°C, washed with 1 ml of ice cold PBS, spun again and resuspended in 500 µl of ice cold PBS. Flow cytometry data was collected on LSRII instrument (Becton Dickinson) and analyzed by Flow Jo software. In each case, around 20,000 GFP-positive events were analyzed.
Hek trex
Manufactured by Thermo Fisher Scientific
The HEK TRex is a laboratory equipment product from Thermo Fisher Scientific. It is designed for the inducible expression of recombinant proteins in HEK293 cells. The core function of the HEK TRex is to provide a versatile and controllable system for the expression of target proteins in a human cell line.
Automatically generated - may contain errors
2 protocols using hek trex
1
Quantifying Polybasic Sequence Stalling in Cells
2
Quantifying Polybasic Sequence Stalling in Cells
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HEK 293T and HEK TRex cells were from Invitrogen and cultured in DMEM (Gibco) containing 10% Fetal Calf Serum (FCS). The cells were routinely tested for mycoplasma and found to be negative. Cells were not authenticated. For the analysis of stalling on polybasic sequences, cells were first plated on 24-well plate and transfected 24 h later with dual-fluorescent reporter constructs using TransIt 293 reagent (Mirus) according to manufacturer’s protocol. After 20 h of expression, cells were washed with PBS and trypsinised. After inhibiting trypsin, cells were collected into 1.5 ml tubes, spun at 5,000 rpm for 3 min at 4°C, washed with 1 ml of ice cold PBS, spun again and resuspended in 500 µl of ice cold PBS. Flow cytometry data was collected on LSRII instrument (Becton Dickinson) and analyzed by Flow Jo software. In each case, around 20,000 GFP-positive events were analyzed.
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