Optiplate 96

For measurement of LDLR promoter activity, HEK293T-ldlrG1 cells were seeded onto a 12-well plate, and after reaching 80% confluence, cells were starved for the indicated time in serum-depleted media supplemented with 0.3% BSA. Luciferase activity was assessed with Luc-Pair™ Duo-Luciferase Assay Kit 2.0 (GeneCopoeia, Rockville, MD, USA), following the manufacturer’s instructions. Briefly, cells were lysed via adding Ly-sis Buffer directly on a 12-well plate (250 µL/well) and incubated for 10 min on a rocking platform. An amount of 20 µL of each cell lysate was transferred in triplicate into the wells of white OptiPlate-96 (Perkin Elmer, Akron, OH, USA), and then 100 µL of FLuc Assay Working Solution was added to each well, mixed and incubated for 3 min. Luminescence was detected via Cytation3 imaging reader (BioTek, Winooski, VT, USA) within 5 min. Luciferase activity was normalized to sample protein concentration and depicted as RLU/µg protein. Results are presented as averages from three independent experiments.

10⁷ protoplasts were co-transformed with 3 µg of the NanoLuc vector and 3 µg of the GFP vector pCop007. Transformed protoplasts were incubated at 28 °C for 16–24 hrs. After incubation, protoplasts were centrifuged for 5 min at 600 g. Pellets were re-suspended in 100 µl of phosphate buffer (pH 5.5), mixed by pipetting, and disrupted by 10 time cycles of the 30 sec-sonication with the Bioruptor sonicator (CosmoBio) with HIGH setting/the 30 sec-cooling down on ice. The cell lysates were transferred to OptiPlate-96 (PerkinElmer), and the Luc activity quantified with a Nano-Glo Luciferase assay kit (Promega) following the manufacturer’s protocol. Luminescence was measured with a luminometer GloMax®-Multi Detection System (Promega). Normalized luminescence of each sample was calculated by dividing the luminescence by the transformation rate as following. $$normalizedluminescence=\frac{\mathrm{Luminascence}\mathrm{measured}\mathrm{by}\mathrm{GloMax}}{\mathrm{Number}\mathrm{of}\mathrm{GFP}\mathrm{positive}\mathrm{cells}\mathrm{in}\mathrm{unit}\mathrm{area}}$$

Luciferase activity of cell lysates was accessed using Bright-Glo™ Luciferase Assay System (Promega; Madison, WI) according to manufacturer's instructions with minor modifications. Briefly, cell lysates (as described previously) or cell suspensions in D-PBS were added to a 96-well white plate (OptiPlate-96, Perkin Elmer), followed by the addition of an equal volume of 2x reconstituted Bright-Glo™ Assay Reagent. Acquisition of the luminescent signal was performed on the Infinite® M1000 PRO microplate reader (Tecan; Männedorf, Switzerland). The mean maximum signal from technical replicates was used to estimate the luciferase activity for each sample. Background readings were measured after incubation of equal volumes of 2x luciferase reagent with either D-PBS or lysis buffer (as appropriate). Background signal was subtracted from sample signal, and normalized either by cell number or protein content per reaction. The ratio of background signal to sample signal (signal-to-noise ratio) was calculated for each sample. Sample signals less than 2-fold above background were considered to not be reliably detectable. To estimate detection threshold for a given sample, the background signal was normalized to cell number or protein amount in the reaction.

Coelenterazine was purchased from Carbosynth. Linear polyethylenimine (PEI, 25 kDa) was supplied by Polyscience. FuGENE® HD Transfection Reagent, furimazine (NanoGlo®), HaloTag® NanoBRET™ 618 ligand and T4 DNA ligase were from Promega. Phosphosite-specific antibodies pS355/pS356-β2 (Cat. no.: 7TM0029A) and pT360/pS364-β2 (Cat. no.: 7TM0029B) were from 7TM Antibodies, and the antibody against pS261 (Cat. no.: PA5-12977) was from Invitrogen. Anti-HA-tag antibodies were purchased from Cell Signaling Technology. Hanks balanced salt solution (HBSS), Dulbecco’s modified Eagles’s medium (DMEM), Lipofectamin 2000, HA-beads, TetraSpeck fluorescent beads, 96-well white polystyrene LumiNunc microplates, all antibiotics and pertussis toxin (PTX) were from Thermo Fisher. The HTRF cAMP accumulation kit and the DERET substrate, SNAP-Lumi4-Tb, were purchased from Cisbio. SNAP-Surface Alexa Flour 488, SNAP-Surface Alexa Flour 549, SNAP-Surface Alexa Flour 649, Q5® High-Fidelity polymerase, NotI-HF®, ApaI, anti-SNAP-tag antibody (Cat. no.: P9310S) were obtained from New England Biolabs (NEB). Effectene was from Qiagen. All other chemicals and compounds were from Sigma Aldrich. TopSeal-A PLUS, Dihydroalprenolol Hydrochloride ([³H]-DHA, 250µCi, 9.25MBq), WGA PVT 500 MG SPA Beads, and OptiPlate-96, and White Opaque 96-well Microplates were from Perkin Elmer.