Anti nf kb

For detection of ALPK1, the cells were lysed with RIPA buffer containing protease inhibitors on ice for 10 minutes. The extracts were centrifuged at 14 500  g for 5 minutes at 4°C. Protein samples were separated by SDS‐PAGE gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies as indicated and peroxidase‐conjugated secondary antibodies and protein signals detected by enhanced chemiluminescence reagent. The primary antibodies used were as follows: anti‐ALPK1 (1000‐fold of dilution; GeneTex Inc), anti‐lectin (1000‐fold of dilution; Bioorbyt), anti‐NFkB (1000‐fold of dilution; Abcam) and anti‐Actin (3000‐fold of dilution; GeneTex Inc).

Western blotting was performed as previously described (Zhang C. et al., 2018 (link)). MCCs were harvested by whole-cell lysates with ice-cold lysis buffer (1% NP-40, 50 mM Tris–HCL, 0.1%SDS, and pH 7.4 150 mM NaCl) and protease inhibitors (Beyotime, Shanghai, China). Normalized by BCA assay kit (Beyotime, Shanghai, China), proteins were separated by SDS-PAGE at 80 V for 30 min, then 120 V for 1 h. After proteins transferred, the PVDF membrane (Millipore, Billerica, MA, United States) was then blocked in 5% non-fat milk at 26°C for 1 h. Next, the blocked membrane was hybridized with primary antibodies anti-Smad3 (1:5,000 dilution; Abcam, Cambridge, MA, United States), anti-TGF-β3 (1:1,000 dilution; Abcam, Cambridge, MA, United States), anti-NF-kB (1:1,000 dilution; Abcam, Cambridge, MA, United States), anti-NF-kB2 (1:1000 dilution; Abcam, Cambridge, MA, United States) and anti-β-actin (1:5,000 dilution; Abcam, Cambridge, MA, United States) at 4°C over night, followed by secondary antibodies goat anti-rabbit IgG (1:5,000 dilution; Abcam, Cambridge, MA, United States) at 26°C for 1 h. Finally, the membrane was incubated with ECL Western blotting Kit (Beyotime, Shanghai, China). The immuno-reactivity percentages to control group was analyzed by Image J or Image Lab software provided by the Institute of Stomatology.

Cells were collected and lysed in M-PER Mammalian Protein Extraction Reagent. All samples were normalized by their protein concentrations and separated in 10% SDS-PAGE gels and then transferred to membranes (Washington, NY) using the wet transfer blotting system (Hercules, CA). The antibodies used for Western blotting were: anti-CD63 (Abcam), anti-TRAF6 (Abcam), anti-TLR4 (Abcam), anti-NF-kB (Abcam), anti-IRKA1 (Abcam), and anti-GAPDH (Abcam), at a dilution of 1:1000. Then, protein bands were developed using ECL reagents, whose images were acquired using the ChemiDoc Imaging system. This western blot examination was repeated three times.

The tissues were electrophoresed on 12% SDS Page gel and then transferred to PVDF transfer plates. The plates were blocked with 5% skimmed milk with 1% Tween in PBS for one hour at room temperature and then incubated for one night by anti-TLR4 and anti-NF-KB purchased from Abcam Company. The effect was measured by Amercontrol software.

Paraffin-embedded tumor tissue sections were cut. After dewaxing and rehydration, sections were treated with antigen retrieval solution for 15 min at 100 °C. Subsequently, samples were blocked with 5 % BSA in PBST for 30 min. Then slides were incubated with anti-TDO2 (1:200, Abcam, UK), anti-IDO1 (1:200, Abcam, UK), anti-IL-6 (1:200, Abcam, UK), anti-Kyn (1:200, Abcam, UK), anti-pSTAT3 (1:200, Abcam, UK), anti-NF-kB (1:200, Abcam, UK) antibodies overnight at 4 °C. After that, samples were incubated with HRP-conjugated secondary antibody (1:1000, Thermo, USA), developed by 3,3-diaminobenzidine solution and counterstained with hematoxylin. The intensity of protein expression in immunohistochemistry was determined by Image-Pro Plus 6.0 software. 8 tumor tissues from 8 patients were included in each group. 10 fields in each tumor tissue were analyzed and the mean of 10 values was determined as the expression intensity of this tumor tissues.

The following commercial primary antibodies were used: 1:1,000 anti‐phospho‐NF‐κB (Cell Signaling Technology, Danvers, MA, USA); 1:1,000 anti‐NF‐kB and 1:1,000 anti‐VCAM1 (Abcam, Cambridge, United Kingdom); 1:200 anti‐paxillin (Santa Cruz Biotechnology, Heidelberg, Germany); and 1:4,000 anti‐β‐actin (Sigma‐Aldrich, St Louis, MO, USA). Secondary antibodies were as follows: 1:1,000 anti‐rabbit IgG Horseradish peroxidase linked F(ab’)2 I fragment (from donkey) (GE Healthcare, GE, Little Chalfont, United Kingdom); 1:3,000 anti‐mouse IgG1 (BD Pharmingen, Beckton Dickinson, Franklin Lakes, NJ, USA); and 1:400 Alexa Fluor 488 conjugated anti‐mouse (from donkey) (Thermo Fisher Scientific, Rockford, IL, USA).