P y705 stat3

Wogonin was isolated from S. baicalensis Georgi according to previous protocols35 (link). Wogonin was of ≥99% or higher in all experiments, unless otherwise noted. Wogonin was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (100 mM), stored at −20 °C, and diluted to each of the designated concentrations in the buffer solution before each experiment. The final concentration of DMSO did not exceed 0.1%. ADR were purchased from ZheJiang HiSun Pharmacetuical Co., Ltd. (Zhejiang, China). IM was purchased from Melonepharma (Dalian, China). Primary antibodies of β-actin (1:2000), NF-κB (1:500), p-IKKα (1:500), IKKα (1:500), IκBα (1:500) and p-IκBα (1:500) were obtained from Santa Cruz (Santa Cruz, CA). Nrf2 (1:1000), p-ERK (1:1000), Tubulin (1:1000), Stat3 (1:1000), pY705-Stat3 (1:1000) and Lamin A (1:1000) were from Bioworld (OH, USA). RPMI-1640 (Gibico, Carlsbad, CA) and DAPI (Invitrogen, USA) were purchased. The IRDye^TM 800 conjugated secondary antibodies were the products of Rockland Inc. (Philadelphia, PA). FITC-conjugated anti-human CD13 antibody was purchased from eBioscience. Epidermal growth factor (EGF) was purchased from Sigma, USA.

Endometrial tissues were fixed in 10% neutral-buffered formalin for 24 h, routinely processed and embedded in paraffin. Tissue sections were immunostained with primary antibodies against HOXA10 (1:50; Abcam, Cambridge, CA, USA), IL-10 (1:1000; Abcam), STAT3 (1:1000; Abcam), or p(Y705)-STAT3 (1:1000; Bioworld) overnight at 4°C, followed by incubation with rabbit anti-goat IgG or goat anti-rabbit IgG and an avidin-biotin complex (Boster Biological Technology, Wuhan, Hunan, China) for 1 h each at room temperature. Finally, the sections were stained with 3,3-diaminobenzidine (DAB) and counterstained with hematoxylin. Control sections were run concurrently with the experimental sections using nonspecific goat IgG and rabbit IgG, and they were similarly pretreated. Nonspecific staining was not detected in the controls. Quantitative analysis was performed using the Image Pro Plus System 6.0 (Media Cybernetics, Inc., Silver Spring, MD, USA) in a blinded fashion, without knowledge of the tissue source. The representative objective protein staining intensity (indicating the relative expression level) was determined according to the mean and integrated optical density (IOD) of the digital image (×400) according to the software's instructions. Signal density data for the tissue areas were obtained from five randomly selected fields of view and subjected to statistical analysis.

Tissues and cells were homogenized in whole-cell lysis buffer (50 mM Tris-HCl [pH 7.6], 150 mM NaCl and 1.0% NP-40) containing phosphatase and protease inhibitors. Immunoblotting was performed with primary antibodies against HOXA10 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), IL-10 (1:1000; Bioworld, St. Louis Park, MN, USA), STAT3 (1:1000; Cell Signaling Technology, Danvers, MA, USA), p(Y705)-STAT3 (1:1000; Bioworld), or GAPDH (1:10,000; Bioworld), followed by a donkey anti-goat or goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (HRP). Detection was performed using an enhanced chemiluminescence kit (Millipore, Billerica, MA, USA).