Journal: Assay and Drug Development Technologies

Article Title: Development of a Time-Resolved Fluorescence Resonance Energy Transfer Ultrahigh-Throughput Screening Assay for Targeting the NSD3 and MYC Interaction

doi: 10.1089/adt.2017.835

Figure Lengend Snippet: TR-FRET assay development for NSD3/MYC interaction. (A) Selection of constructs and TR-FRET antibodies for NSD3/MYC TR-FRET assay development. HEK293T cells were seeded into a 384-well plate and transfected with various tagged DNA constructs. After 48 h, TR-FRET assay was carried out directly in the plate as described in “Materials and Methods” section. A total of 48 combinations were tested as indicated in Table 1. S/B ratio was calculated for each condition. The data are presented as mean ± SD from triplicate samples. (B) The principle of our selected system for NSD3/MYC TR-FRET assay development. In cell lysate with overexpression proteins, VF-NSD3 protein was labeled with Tb through anti-Flag-Tb (N) to serve as a TR-FRET donor. GST-MYC (M) was coupled to TR-FRET acceptor d2 using anti-GST-d2. Interaction between NSD3 and MYC brings Tb and d2 into proximity, leading to the generation of TR-FRET signals. (C) TR-FRET signal from titration of stock cell lysate in a 384-well format. Transfection was performed in a six-well plate and stock cell lysate was collected after 48 h. Increasing amount of stock cell lysate with overexpressing VF-NSD3 and GST-MYC (15 μL) was mixed with diluted anti-Flag-Tb and anti-GST-d2 antibodies (15 μL). The TR-FRET signal was measured. The data shown are average from triplicate samples with SD. GST, glutathione S-transferase; NSD3, nuclear receptor binding SET domain protein 3; S/B, signal-to-background; SD, standard deviation; Tb, terbium; TR-FRET, time-resolved fluorescence resonance energy transfer; VF, Venus-Flag. Color images available online at www.liebertpub.com/adt

Article Snippet: Reagents Tb cryptate-conjugated mouse monoclonal anti-Flag antibody (anti-Flag-Tb, #61FG2TLB) and d2-conjugated anti-GST antibody (anti-GST-d2, #61GSTDLB) were purchased from Cisbio Bioassays (Bedford, MA).

Techniques: Selection, Construct, Transfection, Over Expression, Labeling, Titration, Binding Assay, Standard Deviation, Fluorescence, Förster Resonance Energy Transfer

Journal: Assay and Drug Development Technologies

Article Title: Development of a Time-Resolved Fluorescence Resonance Energy Transfer Ultrahigh-Throughput Screening Assay for Targeting the NSD3 and MYC Interaction

doi: 10.1089/adt.2017.835

Figure Lengend Snippet: Validation of a positive compound, TF-3, in GST pull-down assay. (A) Dose-dependent inhibition of TF-3 on NSD3/MYC interaction in GST pull-down assay. Cell lysates were incubated with increasing concentrations of TF-3 for 2 h at 4°C before performing GST pull-down, and then incubated with GST beads for another 2 h. The amount of VF-NSD3 in the GST-MYC complex was detected by Western blotting with antibodies against Flag and GST. (B) Quantification of the Western blot data in (A). The amount of Flag-NSD3 in the GST-MYC complex was normalized with signals of GST-MYC. Data are presented as mean ± SD (n = 3).

Article Snippet: Reagents Tb cryptate-conjugated mouse monoclonal anti-Flag antibody (anti-Flag-Tb, #61FG2TLB) and d2-conjugated anti-GST antibody (anti-GST-d2, #61GSTDLB) were purchased from Cisbio Bioassays (Bedford, MA).

Techniques: Pull Down Assay, Inhibition, Incubation, Western Blot