Sre luc reporter

4.5 × 10⁴ HEK293A cells were seeded in poly-D-lysine coated 96-well plates (Greiner 655983). Cells were transfected with the following plasmids and amounts per well: 25 ng SRE-Luc reporter (E134A, Promega), 75 ng Gα_i or Gα_i QL in pcDNA3.1+, 2.5 ng cmyc-PRG unless otherwise indicated. Minor adjustments in added DNA were made to equalize expression of Gα_i subunits based on western blotting of Flag tagged constructs. In these cases, empty pcDNA3.1+ vector supplemented to equalize total DNA added per well. Transfection took place immediately after seeding with a 1:3 mass to volume ratio of DNA to Lipofectamine 2000 (Invitrogen). Twelve hours after transfection, the media was replaced with 75 μL of serum-free media. Twenty-four hours after transfection, 75 μL (1:1 volume) of One-Glo reagent (E6110, Promega) was added to each well and incubated for 10 min at room temperature. The luminescence signal was measured using Varioskan LUX multimode microplate reader (Thermo Fisher Scientific).

A total of 4.5 × 10⁴ HEK293A cells were seeded in poly-D-lysine coated 96-well plates (Greiner Bio-One 655983). Cells were transfected with the following plasmids and amounts per well: 25 ng SRE-Luc reporter (E134A, Promega), 75 ng Gα_i or Gα_i QL in pcDNA3.1+, 2.5 ng cmyc-PRG unless otherwise indicated. Minor adjustments in added DNA were made to equalize expression of Gα_i subunits based on Western blotting of Flag tagged constructs. In these cases, empty pcDNA3.1+ vector supplemented to equalize total DNA added per well. Transfection took place immediately after seeding with a 1:3 mass to volume ratio of DNA to Lipofectamine 2000 (Invitrogen). Twelve hours after transfection, the media were replaced with 75 μl of serum-free media. Twenty-four hours after transfection, 75 μl (1:1 volume) of One-Glo reagent (E6110, Promega) was added to each well and incubated for 10 min at RT. The luminescence signal was measured using Varioskan LUX multimode microplate reader (Thermo Fisher Scientific).

The SRE-Luc reporter assay was also performed nearly identically in 24-well plates, which offered better well-to-well consistency for technical replicates. 1 × 10⁵ HEK293A cells were seeded in poly-D-lysine coated 24-well plates. One hundred ng SRE-Luc reporter (E134A, Promega), 300 ng Gα_i or Gα_i QL in pcDNA3.1+, and 5 ng cmyc-PRG DNA were transfected into each well except in Gα_i titration experiments, where reduced Gα_i DNA was substituted with empty pcDNA3.1+. Transfection took place immediately after seeding with a 1:3 mass to volume ratio of DNA to Lipofectamine 2000 (Invitrogen). Twelve hours after transfection, the media was replaced with 250 μL of serum-free media. Twenty-four hours after transfection, 250 μL (1:1 volume) of One-Glo reagent (E6110, Promega) was added to each well and incubated for 10 min at room temperature. The luminescence signal was measured using Varioskan LUX multimode microplate reader (Thermo Fisher Scientific). We found that the fold differences in activation by Gα_i were lower in the 24 well format but that the technical replicates were more reliable.