Anti phospho p65

Total protein was extracted using 2% SDS lysis buffer including protein phosphatase inhibitor (Applygen, Beijing) and heated for 10 min. Protein samples were separated by 8% (v/v) SDS-PAGE gels and transferred onto nitrocellulose membranes (Millipore, Ireland). After blocking in 5% (w/v) Albumin Bovine-V (BSA-V, Solarbio, China) for 1 h at room temperature, the protein bands were incubated with primary antibodies at a dilution ratio of 1:1000 overnight at 4 °C. The primary antibodies contain anti-GAPDH (FL-335; Santa Cruz Bio technology), anti-TGM2 (Proteintech), anti-PD-L1 (Proteintech), anti-STAT3 (CST) and anti-Phospho-STAT3 (Ser705, CST), anti-Akt (CST), and anti-Phospho-Akt (Ser473, CST), anti-P65 (Abcam) and anti-Phospho-P65 (Ser536, Abcam). The protein bands were incubated in HRP-conjugated secondary antibodies (Zsbio, China, 1:5000) at room temperature for 1 h.

FaDu cells grown in 6-well plates were treated with pepsin (0.2 mg/mL) in pH7.0 for 30 min. Levels of phosphorylated of IκB and p65 were evaluated by Western analysis. Rabbit polyclonal anti- p65, anti-phospho-p65, anti-IκB, and anti-phospho- IκB antibodies were purchased from Abcam (Cambridge, UK). The secondary antibodies were Goat anti-rabbit antibodies conjugated with horseradish peroxidase purchased from Abcam (Cambridge, UK). Signals were visualized by ChemiDoc XRS+ using the Image LabTM Software (Bio-Rad Laboratories, Munich, Germany). Protein levels were quantified by scanning densitometry.

Protein samples were extracted with RIPA-buffer (Heart, China) from LTP-treated cells after 24 h in culture, then heated at 95 °C °C for 5 min with sample buffer (250 mM Tris-HCl, pH 6.8, 10% glycerol, 4% sodium dodecyl sulfate, 2% β-mercaptoethanol, and 0.003% bromophenol blue). Proteins were separated on 12% SDS-PAGE, and electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, USA), and then blocked with 5% dried skim milk in Tris buffer saline with 0.1% Tween-20 (TBST) for 2 h at room temperature. The blocked membranes are rinsed three times in TBST for 10 min, and then incubated in the primary antibodies (anti-phospho p65, 1:5000, Abcam; anti-NF-κB P65 and anti-NF-κBIα Polyclonal Antibody, 1:2000, ABclonal; anti-cyclinD1, 1:200, Santa Cruz; anti-β-actin, 1:2000, CWbio) overnight at 4 °C. After being washed three times with TBST, the secondary antibody horseradish peroxidase (CWbio) was incubated on the membrane for 2 h at room temperature. Then the PVDF membranes were washed three times with TBST and incubated with ECL Plus Reagent (Millipore, USA) and scanned using the chemiluminescence (BioRad, USA). Immunoreactive products were quantified by image-Pro plus 6.0 software to determine the optical density of protein bands.