Recombinant human her2 erbb2 protein

The TCM compounds’ affinity for proteins was measured by MST Reagent (NanoTemper Technologies) and Monolith NT.115 (NanoTemper Technologies). The proteins (Human PD‐L1 Fc Protein, PeproTech; and Recombinant Human Her2/ErbB2 Protein, Sino Biological) were desalted into MST buffer (10 mmol/L HEPES, pH 7.5, 150 mmol/L NaCl) before the experiment. The proteins were fluorescently labelled and the protein concentration was adjusted to 10 μmol/L. Fluorescent dye NT‐647‐NHS was added, mixed, and incubated for 30 min at 25°C in the dark. For each assay, the labelled protein (approximately 0.1 μmol/L) was mixed with the same volume of unlabeled ligands quercetin (Sigma‐Aldrich), daidzein (Sigma‐Aldrich), isorhamnetin (Sigma‐Aldrich), or formononetin (Saitong) in 16 serial concentrations at room temperature. The samples were then loaded into premium capillaries and measured at 25°C by using 20% Light Emitting Diode power and medium MST power. Data analyses were undertaken using MO Affinity Analysis 2.2.4 software. With a confidence of 68%, the K_d value is within the given range.

Recombinant human Her-2/ErbB2 protein (cat# 10004-H20B1) was purchased from Sino
Biological Inc. ER, PR, and Ki67 recombinant proteins were purified in the
house. QDB plate was purchased from Quanticision Diagnostics, Inc (RTP). Anti-PR
(clone 1E2, cat# 790-4296) rabbit monoclonal primary antibody was purchased from
Roche Diagnostics GmbH. Rabbit anti-ER (clone SP1, cat# ab13370) antibody was
purchased from Abcam Inc. Rabbit anti-Her2 antibody (clone EP3, cat# ZA-0023)
and mouse anti-Ki67 (clone MIB1, cat# ZM-0167) were purchased from ZSGB-BIO
(www.zsbio.com). HRP labeled Donkey Anti-Rabbit IgG secondary
antibody was purchased from Jackson Immunoresearch lab (West Grove). BCA total
protein quantification kit was purchased from Thermo Fisher Scientific Inc
(Calsband).