Phospho stat3 ser727

Sections were cut (10 µm) from paraffin embedded tissues and stained with hematoxylin–eosin reagent or incubated with primary antibodies as indicated. The following primary antibodies were used for immunohistochemistry (IHC): phospho-stat3 (Tyr705) (Cell Signaling Technology), and phospho-stat3 (Ser727) (Abcam). Five representative images from each section were taken at 400× magnification using bright field microscopy (Leica DMi8). For each image, positive staining positive nuclei were counted using ImageJ software (National Institutes of Health; Bethesda, MD, USA). Positively staining cells are presented as a percentage of total number of cells counted.

Protein lysates from cancerous and normal lung tissues as well as 16HBE cells were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (BioTrace^TM, Pall Corporation, San Diego, CA, USA). After blocking with 5% skim milk in phosphate-buffered saline (PBS), the membranes were incubated with a primary antibody against the selected key proteins, including NF-κB p65 or phospho-NF-κB p65 (Ser536) (1:1000; Abcam, Cambridge, UK), STAT3 or phospho-STAT3 (Ser727) (1:1000; Abcam), cyclinD1 (1:1000; Abcam), β-actin (1:5000; Proteintech Group Inc., Chicago, IL, USA) or Lamin B1 (1:5000; Abcam), and the bound antibodies were detected by using a Super Signal West Pico Kit (Thermo Fisher Scientific Inc., USA), followed by quantitative densitometric analysis using Eagle Eye II software.

The cells were harvested, washed, and lysed with RIPA lysis buffer containing a protease inhibitor cocktail (BBI Life Sciences, Shanghai, China) together with NaF and Na₄P₂O₇, and the protein concentration was measured using a BCA Protein Assay Kit (Pierce, Waltham, MA, USA). Subsequently, the samples were loaded onto SDS-PAGE gels and separated through electrophoresis. The proteins were transferred onto PVDF membranes and incubated with the corresponding primary Abs and HRP-conjugated secondary Abs. Chemiluminescent detection was performed using Alliance 4.7 (UVITEC Cambridge, Cambridge, UK) with Luminata Forte Western HRP Substrate (Millipore, Billerica, MA, USA).
The following antibodies were used: Phospho-IκBα (Ser32) (CST#2859, Cell Signaling Technology, Danvers, MA, USA), IκBα (CST#4812), Phospho-NF-κB p65 (Ser536) (CST#3033), NF-κB p65 (CST#3034), NIK (CST#4994), NF-κB2 p100/p52 (CST#4882), RelB (CST#4922), Phospho-Stat3 (Tyr705) (CST#9131), Phospho-Stat3 (Ser727) (CST#9134), SHP-1 (CST#3759), SHP-2 (CST#3397), Stat-3 (ab68153, Abcam, Cambridge, UK), β-Actin (ACTB) (BM0627, Boster, Wuhan, China), HRP Anti-rabbit IgG (CST#7074), and HRP anti-mouse IgG (BioLegend, 405306).