Mouse anti iκbα antibody

2-Octen-1-ylsuccinic anhydride, RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin, polymyxin B sulfate salt, and LPS from Escherichia coli 026/B6 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA) and Biolegend (San Diego, CA, USA), respectively. Goat anti-actin antibody and anti-goat IgG antibody labeled with horseradish peroxidase (HRP) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse anti-IκBα antibody, HRP-labeled anti-mouse IgG antibody, HRP-labeled anti-rabbit IgG antibody, and rabbit antibodies against histone H3, NF-κB p65, ERK1/2, phosphorylated ERK1/2, JNK, phosphorylated JNK, p38 MAPK, and phosphorylated p38 MAPK were purchased from Cell Signaling Technology (Danvers, MA, USA). TAK-242 was purchased from Chemscene (Monmouth Junction, NJ, USA).

50 µl of the concentrated culture medium or 100 µg protein were boiled in Laemmli buffer for 5 min. The samples together with biotinylated protein ladder (Cell Signaling, Danvers, MA, USA) were separated on 13% SDS-PAGE and blotted onto nitrocellulose membranes using a mini transblot cell (BioRad) in blotting buffer (25 mM TRIS, 192 mM glycine, 20% methanol) for 90 min at 100 V. The blots were blocked using Roti-Block (Roth, Karlsruhe, Germany) for 1 h at room temperature. Incubations with rabbit anti-VEGF antibody (1∶1000; Santa Cruz, Santa Cruz, CA, USA), mouse anti-IκBα antibody (1∶1000; Cell Signaling), or mouse anti-phospho-IκBα antibody (1∶1000; Cell Signaling) were performed at 4°C overnight. Secondary incubations with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse antibodies, diluted 1∶50,000, were performed for 1 h at RT. The reactions were visualized by chemiluminescence imaging using using Immobilon chemiluminescent HRP-substrate (Millipore, Schwalbach, Germany) with GeneGnome imaging system (Syngene, Cambridge, UK). After stripping using Restore Westernblot stripping buffer (Thermo, Waltham, MA, USA), subsequent incubations with mouse anti-β-actin antibody (Sigma), diluted 1∶10,000, and secondary incubations with anti mouse antibodies, diluted 1∶50,000, were performed accordingly.

The antibodies used were rabbit anti-p44/42 MAPK (Erk1/2) antibody #9102 (Cell Signaling Technology, Beverly, MA), rabbit anti-phospho-Erk1/2 antibody #9101 (Cell Signaling Technology), rabbit anti-phospho-p38 MAPK antibody #4511 (Cell Signaling Technology), rabbit anti-p38 MAPK antibody #8690 (Cell Signaling Technology), rabbit anti-phospho-SAPK/JNK antibody #4668 (Cell Signaling Technology), rabbit anti-SAPK/JNK antibody #9258 (Cell Signaling Technology), mouse anti-phospho-IκBα #9246 (Cell Signaling Technology), mouse anti-IκBα antibody #4814 (Cell Signaling Technology), rabbit anti-LTβR, N-Terminal antibody #SAB4501788 (Sigma-Aldrich), goat anti-LTβR antibody #L5412 (Sigma-Aldrich), normal goat IgG control #AB-108-C (R&D systems), anti-mouse-IgG HRP-linked antibody #7076 (Cell Signaling Technology), and anti-rabbit IgG-HRP-linked antibody #7074 (Cell Signaling Technology).
Equal protein loading was confirmed by probing the blot with mouse anti-α-tubulin (Sigma-Aldrich) antibody.