Rabbit anti zo 1

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.

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.