Steadylite

10⁶293 T cells were transfected with 300 ng TRIM21-His (WT or S80A) and 3 μg empty vector or IKKβ (S177E/S181E). 48 hr later cells were washed, pelleted, denatured in 500 μL 6 M GuHCl, 0.1 M Na2HPO4/NaH2PO4 (pH 8), 10 mM Imidazole (pH 8) and samples rotated for 3 hr at room temperature with 30 μL equilibrated NiNTA agarose (Qiagen). The agarose matrix was washed twice with 500 μL lysis buffer, twice with 500 μL 3:1 wash buffer:lysis buffer, once with 500 μL wash buffer (25 mM Tris, 20 mM imidazole pH 6.8), resuspended in 2 × LDS sample buffer supplemented with 300 mM Imidazole to elute bound His-tagged proteins and heated for 5 min at 95°C before LDS-PAGE. For assaying IKKβ activity in the absence of NF-κB signaling, 10⁶293T were transfected with 500 ng pGL4.32[luc2P/NF-κB-RE/Hygro], 200 ng TRIM21-His and 2 μg empty vector or IKKβ. 6 hr later, cells were treated with DMSO or 30 nM bortezomib for 16 hr. Cells were washed, resuspended in 500 μL cold PBS. 20 μL cells were mixed with 100 μL SteadyLite (Perkin Elmer) and luminescence measured (Pherastar). The remaining cells were pelleted, denatured and mixed with NiNTA agarose as described above. For TBK1 and MAVS transfection experiments, 200 ng TRIM21-His was transfected with 2 μg empty vector or TBK1 or MAVS. Cells were harvested at 24 hr post transfection.

The antiviral activity of the compounds was determined by pre-incubating 10⁴ TZMbl cells/well in a 96-well plate for 2 h at 37 °C and 7 % CO₂ to maintain the optimal pH with or without a serial dilution of compounds. Next, 200 TCID₅₀ of HIV-1 BaL virus was added to each well, and cultures were incubated for 48 h before luciferase activity was quantified. To this end, 120 μL of supernatant was removed, 75 μl of the luciferase substrate Steadylite (Perkin Elmer, Life Sciences, Zaventem, Belgium) was added to the wells, and the plates were incubated at room temperature on an orbital shaker for 10 min. Next, the luciferase activity was measured using a TriStar LB941 luminometer (Berthold Technologies GmbH & Co. KG., Bad Wildbad, Germany) and expressed in relative light units (RLU).
Each compound was tested in triplicate and each experiment was repeated in three independent runs. Antiviral activity was expressed as the percentage of viral growth compared to the control and plotted against the compound concentration. Next, non-linear regression analysis was used to calculate the EC50_, using GraphPad Prism 5.03 using non-linear regression (GraphPad Software, San Diego, CA, USA).

Recombinant hCG and LH were purchased from National Peptides and Hormones Program (c/o A. F. Parlow, Harbor-UCLA Medical Center). For PALM studies, CAGE 500 and 552 N-hydroxysuccinimide esters for antibody conjugation and direct labeling of receptors were purchased from Abberior. Primary antibodies HA.11 and FLAG were purchased from Covance and Sigma, respectively. For BRET and pRL-cmv luciferase reporter assays, coelentrazine h and coelentrazine, respectively, were purchased from Promega. For cre-luc reporter gene assays, SteadyLite was purchased from PerkinElmer Life Sciences. Fluo-4 direct for calcium imaging was obtained from Invitrogen and HTRF-IP₁ assay from CisBio.

Luciferase assays were performed on HEK-293 cells or CRISPR/Cas9 genetically engineered HEK-293A cells depleted of different G proteins and parental HEK-293A cells. Cells were seeded at 35,000 cells per well on white 96-well plates pre-treated with poly-D-lysine. The following day, cells were transiently transfected with receptor constructs at indicated concentrations and co-transfected with transcription reporter plasmids CRE-Luc, NFAT-Luc or NF-κB-Luc at 30ng/well using Lipofectamine 2000 (Invitrogen). Additional co-transfection with Gα_Δ6qi4myr, which is recognized by GPCRs as a Gα_i protein but elicits Gα_q-dependent signaling (58 (link)) was performed in CRE and co-transfection with Gα_q or Gα₁₁ was performed in NFAT luciferase assay. After 24 hours, cells were washed with PBS and incubated for 30 minutes with a mixture of SteadyLite (50μL/well, PerkinElmer) and PBS (50μL/well). Plates were read on an EnVision Multilabel Plate Reader (PerkinElmer) using the luciferase program. The experiment was performed three times in triplicates.

VSVG HIV-Luc-pseudotyped HIV-1 virus was prepared as described previously [17 (link)]. CEM T cells were infected with the VSVG HIV-Luc virus and incubated overnight. The infected cells were then treated with either PP1-targeting compounds or nanoparticles and incubated overnight. Both uninfected cells and cells infected with the pseudotyped virus were used as negative and positive controls, respectively. The luciferase lysis buffer (SteadyLite, Perkin Elmer) was added to the cells and luminescence was measured using the Luminoskan (Perkin-Elmer).

To assess ligand activities on full length MR, U2-OS cells (ATCC HTB96^™) were transiently transfected via electroporation with hMR/pCDNA 3.1 or hMR(M777V)/pCDNA3.1 expression plasmids and a MMTV-luc2P/pGL4.36 reporter plasmid. To determine species specific ligand activities, a reporter plasmid where luciferase is driven by the yeast GAL4 response element was cotransfected with plasmids expressing a fusion protein between the GAL4 DNA binding domain and the human, rat or mouse MR LBD. Transfected cells were seeded in 384 well plates prior to addition of AZD9977 or eplerenone diluted in cell medium (with or without aldosterone) and incubation at 37°C, 5% CO₂ for 24 hours. Cells were lysed with SteadyLite (Perkin Elmer) according to manufacturers protocol and luminescence was measured using an Envision plate reader. Analysis and curve fitting was performed using GraphPad Prism 6.0 (GraphPad Software, Inc, CA, USA).