Phospho iκbα antibody

Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to NC membranes, and blocked with TBST of 5% skim milk for 1 hour. The membranes were washed with TBST and then incubated with the specific primary antibody for 6 hours at 4°C. The membranes were then incubated for 1 hour at room temperature in secondary and the signal was detected by chemiluminescence (Bio rad, USA). The primary (1:1000) and secondary (1:10000) antibodies were purchased from Cell Signaling Technology, USA and listed as follows: GAPDH antibody(#2118), stat3 antibody(#12640), phospho-stat3 antibody(#98543), SAPK/JNK antibody(#9252), phospho-SAPK/JNK antibody(#4668), p65 antibody(#4764), phospho-p65 antibody(#3033), IKKα antibody(#2682), phospho-IKKα/β antibody(#2697), IκBα antibody(#4812), phospho-IκBα antibody(#2859), p38 MAPK antibody(#8690), phospho-p38 MAPK antibody(#4511), Erk1/2 antibody(#4695), phospho-Erk1/2 antibody(#4370) and HRP-linked goat anti-rabbit IgG(#7074).

KN-3 monolayers in 6-well tissue culture plates were stimulated with iE-DAP (10 μg mL⁻¹), iE-Lys (10 μg mL⁻¹), or TNF-α (0.01 μg mL⁻¹) for 5 or 10 min. Stimulated or unstimulated KN-3 cells were collected in RIPA lysis buffer (Santa Cruz Biotechnology). The protein concentrations in lysates were quantified using a bicinchoninic acid protein assay kit (Sigma-Aldrich). Equal amount of protein was loaded onto a 5–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (Bio-Rad Laboratories, Hercules, CA, USA), followed by electrotransfer to a polyvinylidene difluoride membrane. The membrane was first incubated with inhibitor κB (IκB) α antibody (Sigma-Aldrich) or phospho-IκBα antibody (Cell Signaling Technology, Danvers, MA, USA). After washing, the membrane was reacted with horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich). Protein bands were finally visualized on X-ray film with the use of ECL Prime Western Blotting Detection System (GE Healthcare, Buckinghamshire, UK). Actin levels were also assessed using an anti-actin antibody as an internal control (Sigma-Aldrich).

LPS from Escherichia coli (0111:B4), AA and DAPT were purchased from Sigma–Aldrich. Dll4 was obtained from R&D Systems. β-Actin and Hes1 primary antibody were obtained from Abcam, Notch3 and iNOS primary antibody were from Santa Cruz Biotechnology. Phospho-C-Jun N-terminal kinase (JNK) (Thr183/Tyr185) antibody, phospho-extracellular signal-regulated kinase (ERK)1/2 (Thr202/Tyr204) antibody, phospho-p38 (Thr180/Tyr182) antibody, and phospho-IκBα antibody were purchased from Cell Signaling Technology. All secondary antibodies were from Boster Biological Technology. All other reagents were from commercial suppliers and of standard biochemical quality.