Black 384 well plate

Purified bovine β-cardiac myosin was exchanged with mVenus-cRLC as described previously (58 (link)). Native and cRLC-exchanged cardiac myosin were diluted to 0.4 μmol/L in reaction buffer (10 mmol/L PIPES, pH 7, 5 mmol/L MgCl₂, and 1 mmol/L DTT) and incubated on ice for 2 h to form synthetic myosin filaments. 50 μL myosin filament solution per well was aliquoted into a black 384-well plate (GREINER). Mant-ATP was added to a final concentration of 0.8 μmol/L using automated injector units; the system was allowed to age for 90 s, and 2 mmol/L Adenosine-5′-triphosphate (ATP) was added using the automated injectors. Fluorescence intensity was continuously sampled every 1 s for 15 min. Data were fitted to a biexponential decay function extracting the fraction of the slow (SRX state) and fast (DRX state) phases.

Kinetic aggregation studies in the presence of 20 µM ThT were conducted using a BMG Clariostar Plus reader. For this purpose, gN1 and hN1 (30 µM each) were incubated with 5 mM EDTA or 500 µM Zn(II) in the presence or absence of trypsin at a 1:1,000 w/w enzyme-to-protein ratio for 48 h. Each sample (50 µl) was prepared in triplicate on a black 384-well plate (Greiner). The plate was then centrifuged (1,000 g, 2 min, 20 °C) and scanned in fluorescence intensity mode. ThT was excited at 418 nm (20 flashes/well), and fluorescence emission was measured at 490 nm. The data are expressed as normalized fluorescence units (NFUs) calculated by dividing the number of raw fluorescence units (RFUs) of the samples by the mean free fluorophore emission.

Recombinant human Gαo and Gαi1, wild-type or [Q52P] and [Q52R] mutants were brought into the 20 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl by a 10,000-fold buffer exchange on Amicon 10K ultracentrifugation concentrators (Merck, Kenilworth, NJ USA). For measurement, 2 µM of indicated protein was incubated for 30 min in the black 384-well plate (Greiner, Kremsmünster, Austria) and then mixed with an identical volume of 2 µM BODIPY-FL-GTP or 2 µM BODIPY-FL-GTPγS (both from Thermo Fisher Scientific) in the buffer containing 20 mM Tris-HCl buffer (pH 7.5), 150 mM NaCl, 0.2% BSA and 10mM MgCl₂ using the plate reader injector directly before measurement. The kinetics of in vitro G protein binding and/or hydrolysis was measured in the Infinite M200 Pro multiwell reader (Tecan, Männedorf, Switzerland) [28 (link),29 (link)].

Affinity purified coregulators were labelled with FITC (fluorescein isothiocyanate), in a proportion of 500 ul of coregulator/control affinity elution with 50 ul of 20mM FITC at 4°C for 3 h. The probe excess was removed by a desalting column (HiTrap, 5 ml, GE). To evaluate the affinities between coregulators and PPARγ, serial dilutions of purified PPARγ wild-type (wt) or S273A and S273D mutants (200 μM to 6 nM) were performed using the elution buffer of each coregulator (see Protein Expression and Purification section), in three replicates, in black 384-wellplates (Greiner). The coactivator conditions were incubated also with Rosiglitazone (3× molar excess). In order to measure any unspecific interaction, we performed the same experiment with control expressions of non-induced protein extracts. These extracts were incubated in GST and cobalt resins, labeled with FITC in the same proportion (50 ul in 500 ul of extract elution) and the affinity with PPARγ we and mutants were measured. For each fluorescence curve, the mixtures were submitted to fluorescence anisotropy measurements using ClarioStar^® plate reader (BMG) (emission of 520 nm and excitation of 495 nm). Data were analyzed using the software OriginPro 8.6 and Kd were obtained from fluorescence data fitted to binding curves using Hill model.

IP₃ was measured using the HitHunter IP₃ Fluorescence Polarization Assay Kits (DiscoverRx Tech, Fremont, CA, USA). Briefly, 2 × 10⁴ cells in black 384-well plates (Greiner, Germany) were treated with anti-CD3 for the designated times, and the cellular reaction was terminated by adding 0.2 N perchloric acid. The plate was shaken at 650 r.p.m. for 15 min. The IP₃ tracer was subsequently added to each well, and the IP₃ binding protein was added to the plate. The polarized fluorescence from the IP₃ tracer was read on a Microplate Reader (Synergy 4 Hybrid, BioTek, ). The IP₃ concentration was calculated from the IP₃ standard curve.