His tag labeling kit red tris nta 2nd generation

βArr1-3A and Gα_iq chimera constructs containing N-terminal 6x His-tags were labeled with the His-Tag Labeling Kit RED-tris-NTA 2nd Generation (NanoTemper). A constant concentration of either RED-βArr1-3A or RED-Gα_iq chimera was incubated with varying concentrations of NT8-13-bound enNTS₁ΔM4 before being loaded into NanoTemper premium capillaries and assayed via a NanoTemper Monolith NT MST instrument. βArr1-3A experiments consisted of 16 samples in 50 mM Potassium phosphate pH 7.4, 100 mM NaCl containing 25 nM RED-βArr1-3A, 40 µM NT8-13, 40 µM C8-PIP2, 0.03% DDM and a 1:2 dilution series of enNTS₁ΔM4 from 20 μM to 0.61 nM. Gα_iq chimera experiments consisted of 16 samples in 50 mM Potassium phosphate pH 7.4, 100 mM NaCl containing 25 nM RED-Gα_iq chimera, 100 µM NT8-13, 100 µM C8-PIP2, 0.03% DDM, 2 mM MgCl₂, 10 μM GDP, 0.2 units of apyrase and a 1:2 dilution series of enNTS₁ΔM4 from 50 μM to 1.53 nM. Data was collected using 80% LED and 80% MST power in experimental triplicate and at least three technical repeats. Raw data was fit to a Hill function to establish upper and lower asymptote values. These values were then used to normalize the data and subsequently fit to a quadratic binding function using Sigma Plot v15 (Systat Software Inc.).

Molecular docking was performed using AutoDock 4 and AutoDock Vina with the blind docking, and the structure of TEC and 21 STAT3 inhibitors were retrieved from PubChem (https://pubchem.ncbi.nlm.nih.gov/). The 3D protein structures of the STAT3 monomer (PDB ID: 6njs) and aggregate (PDB ID: 6tlc) were downloaded from RSCB Protein Data Bank (https://www.rcsb.org/), and then processed using PyMol and AutoDockTools. The docking results were visualized by Pymol to display the polar contacts. The affinity heatmap was generated by the Heatmap package in R studio software. The microscale thermophoresis (MST) assay was used to detect the direct affinity between the small molecule and protein in vitro. STAT3 protein was purchased from ACROBiosystems (ST3-H5149, ACROBiosystems, Newark, DE 19711, USA) and labeled using the His-Tag Labeling Kit RED-tris-NTA 2nd Generation (MO-L018, NanoTemper Technologies, Munich, Germany). MST analysis was performed in the Monolith NT.115 system (NanoTemper Technologies) according to the manufacturer’s manual. Results were normalized and analyzed in the Monolith NT.115 system.

To determine KMT9/compound binding affinities, MST was performed using a Monolith NT.115 instrument (NanoTemper Technologies GmbH). His-tagged KMT9 was labelled using the His-Tag Labeling Kit RED-tris-NTA 2^nd Generation (NanoTemper Technologies GmbH) according to the manufacturer’s instructions. Labelling and binding assays were performed in buffer containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 1 mM DTT, and 0.05% Tween. Compound 1:1 dilution series were prepared, mixed with labelled KMT9 protein (20 nM final concentration), and incubated for 30 min at room temperature. After centrifugation, samples were loaded into standard capillaries (NanoTemper Technologies GmbH), and MST measurements were performed at 40% MST and 100% LED power using the MO.Control program. Datasets were processed with the MO. Affinity analysis software (NanoTemper Technologies GmbH).