Mouse anti traf6

Protein was extracted from intestinal tissues and cells using RIPA buffer containing protease inhibitor cocktail and measured using a BCA protein assay kit. An equivalent of protein samples was separated by 12% SDS-PAGE and transferred to PVDF membranes for western blot analysis. After blocking with 5% skimmed milk for 1 h, the membranes were probed overnight at 4 °C with the following different primary antibodies: rabbit anti-GFAP (1:5000, GeneTex, Irvine, CA, USA), rabbit anti-CD40 (1:1000, Abcam, Cambridge, UK), rabbit anti-ZO-1 (1:1000, Affinity, USA), rabbit anti-occludin (1:5000, ABclonal, Wuhan, China), mouse anti-TRAF1 (1:100, Santa Cruz, CA), mouse anti-TRAF2 (1:100, Santa Cruz, CA), mouse anti-TRAF3 (1:100, Santa Cruz, CA), mouse anti-TRAF4 (1:100, Santa Cruz, CA), mouse anti-TRAF5 (1:100, Santa Cruz, CA), mouse anti-TRAF6 (1:100, Santa Cruz, CA), and rabbit anti-GAPDH (1:5000, ABclonal). Then, the membranes were incubated with secondary antibodies at room temperature for 1 h. The immunoreactive bands were detected using a chemiluminescence imaging system ChemiScope 6000 (Clinx, Shanghai, China), and the intensity was analyzed with ImageJ.

All common chemicals were obtained from Sigma-Aldrich (Wicklow, Ireland) unless otherwise stated. Antibodies used for Western blot and co-immunoprecipitation include rabbit anti-Bid (Abcam, ab62469, 1:1000), rabbit anti-Peli1 (Abcam ab199336, 1:1000), rabbit anti-TRAF3 (Abcam, ab76147, 1:500), mouse anti-TRAF6 (Santa Cruz, sc8409, 1:200), rabbit anti-phospho-IRF3 (Cell Signaling, 4947P, 1:500), rabbit anti-phospho-TBK1 (Cell Signaling, 5483P, 1:500), rabbit anti-phospho c-Jun (Cell Signaling, 9261S, 1:1000), rabbit anti-A20/TNFAIP3 (Cell Signaling, 5630, 1:1000), rabbit anti-LC3 (Abcam, ab51520, 1:2000), rabbit anti-FLAG (OctA-Probe D-8, Santa Cruz, sc-807, 1:500), anti- α-Tubulin (Sigma-Aldrich, T6199, 1:5000), anti-β-Actin (Sigma-Aldrich, A3853, 1:5000), and anti-GAPDH (Abcam, ab8245-100,1:5000). Bortezomib was obtained from Millennium Pharmaceuticals (Cambridge, MA, USA). Lipopolysaccharide (LPS) was obtained from (Sigma-Aldrich, L4391), and PolyI:C (31852-29-6) and Pam₃CSK4 (112208-00-1) were obtained from InvivoGen.