Synergy h4 multi mode reader

Fluorescence polarization was measured in 384-well plates (Corning) using Synergy H4 multi-mode reader (BioTek). For direct titration experiments, 50 nM reporter peptide (RSK1_729–735) was mixed with increasing amount of MBP-PDZ domains. In competitive measurements, the 50 nM reporter peptide was mixed with the PDZ domain in a concentration to achieve high degree of complex formation. Subsequently, increasing amount of unlabeled peptide (RSK1_683–735) was added to the reaction mixture. Titration experiments were carried out in triplicate and the average FP signal was used for fitting the data to a quadratic or competitive binding equation.

Ethoxyresorufin-O-deethylation (EROD) activity was measured in both MCF-10A cells and with recombinant proteins as previously described for enzyme modulation and inhibition.^{42 (link),43 (link)} For enzyme modulation, MCF-10A cells were plated in 96-well plates for 24 h. Compounds were added 1 h prior to TCDD (10 nM) for 48 h. Cells were washed twice with PBS and incubated with ethoxyresorufin (2.5 μM) and salicylamide (1.5 mM) at 37°C. For enzyme inhibition, MCF-10A cells pretreated with TCDD for two days were washed with PBS and pre-incubated with test compounds for 5 min. Subsequently, ethoxyresorufin (2.5 μM) and salicylamide (1.5 mM) were added at 37 °C. For the recombinant protein inhibition assay, 0.15 pmole of CYP1A1 or 0.8 pmole of CYP1B1 (BD Biosciences, Woburn, MA) per well was pre-incubated with test compounds or 2,3′,4,5′-tetramethoxystilbene (TMS) for 5 min at 37 °C in 50 mM potassium phosphate buffer (pH = 7.4) with 1 mM NADPH before adding ethoxyresorufin (2.5 μM). EROD activity was measured as resorufin formation by fluorescence with excitation at 530 nm and emission at 590 nm every minute for 20 min at 37 °C using BioTek’s Synergy H4 Multi-Mode reader (Winooski, VT). A resorufin standard curve was used to calculate the P450 1A/1B activity.