Anti phospho ikkα β ser176 180

Cells were lysed in ice-cold lysis buffer with a protease inhibitor cocktail (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min and then centrifuged at 13,300 rpm for 20 min. Thirty micrograms of protein were separated on an SDS-PAGE gel and then transferred. After blocking, the membrane was incubated overnight at 4 °C with a primary antibody including anti-phospho-NF-κB p65 (Ser536), anti-NF-κB, anti-phospho-IκB-α (Ser32), anti-IκB-α, anti-phospho-IKKα/β (Ser176/180) (Cell Signaling Technology, Boston, MA), anti-phospho-IRE1α (Abcam, Cambridge, MA), anti-IRE1α (Abcam, Cambridge, MA), or anti-β-actin (Cell Signaling Technology) antibodies. After incubation with an HRP-conjugated secondary antibody, signals were detected with a chemiluminescence western blotting detection solution using Bio Imaging System (Syngene, Frederick, MD).

The following antibodies were used for western blot: anti-NAMPT (A300-779A, Bethyl Laboratories, Montgomery, TX), anti-NAPRT1 (NBP1-87243, Novus Biologicals, Littleton, CO; AMAB90725 Atlas; 66159-I-Ig ProteinTech, Manchester, UK and MBS1491066 MyBioSource), anti-phospho-p65 (Ser536, #3033S), anti-p65 (#8242S), anti-phospho-IKKα/β (Ser176/180, #2697), anti-IRAK1 (#4504), anti-NLRP3 (#13158), anti-Caspase-1 (#3866) and Cyclophilin A (#2175) all from Cell Signaling Technologies (Danvers, MA), anti-pERK1/2 (pT202/Y204, 612358) and anti-panERK (610123) both from BD Biosciences (East Rutheford, NJ), anti-MyD88 (sc-11356, Santa Cruz Biotechnology, Dallas, TX) and anti-actin horseradish peroxidase (HRP)-conjugated (ab20272, Abcam, Cambridge, UK). Secondary reagents were: goat anti-mouse IgG-HRP-conjugated (Perkin Elmer, Waltham, MA), goat anti-rabbit HRP-conjugated (Santa Cruz Biotechnology).

Cells were washed twice with PBS and lysed on ice with RIPA lysis buffer containing a protease inhibitor and phosphatase inhibitor. The supernatant was collected after the cell lysate was centrifuged, and the protein concentration was quantified using a BCA protein detection kit. Equal amounts of protein were subjected to SDS-PAGE. After separation, the proteins were transferred onto a PVDF membrane and incubated with a specific primary antibody at 4°C overnight. Then, the membrane was incubated with an HRP-conjugated secondary antibody for 1 h. Immunoreactions were detected with a chemiluminescence reagent (Milliwell, WBKLS0050) and analyzed with Image Lab software. The primary antibodies used in this experiment were as follows: anti-VRK2 (Proteintech, 12946-1-AP, 1:1 000), anti-tubulin (Santa Cruz Biotechnology, sc-5286, 1:4 000), anti-Histone H3 (Cell Signaling Technology, 97733s, 1:1 000), anti-Flag (Sigma, F9291; 1:3 000), anti-HA (Sigma, H3663, 1:2 000), anti-P65 (Cell Signaling Technology, 8242s, 1:1 000), anti-IKKβ (Cell Signaling Technology, 2678s, 1:1 000), anti-Phospho-IKKα/β (Ser176/180) (Cell Signaling Technology, 2697s, 1:1 000), and anti-GST (Cell Signaling Technology, 2624s, 1:5 000).