Phospho nf κb p65 ser 536

Nuclear and cytoplasmic protein was extracted with a nuclear and cytoplasmic extract kit (Cayman Chemical). Protein content of each sample was determined using the BioRad protein assay (BioRad Laboratories) according to manufacturer's protocol. For immunoblot analysis, 25 μg of protein was subjected to SDS-PAGE, transferred to a membrane, and probed with specific antibodies: NF-κB p65 (1 : 5000, Abcam); phospho-NF-κB p65 (Ser 536, 1 : 5000, Abcam); Lamin B (1 : 2000, Abcam); β-actin (1 : 2000, Abcam). Secondary antibody HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, 1 : 5000) were used to detect binding of antibodies. The membrane was incubated with Clarity Western ECL Substrate (Bio-Rad Laboratories, CA, USA), and the target proteins were then visualized and quantitated using a LAS-3000 luminescent image analyzer (Fujifilm). The results were expressed as a relative ratio of the target protein to reference protein.

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).