Phospho p65 p p65

Antibodies against p65 (cat.no. 4764), phospho-p65 (p-p65, cat.no. 3033), IκBα (cat.no. 8635), phospho-IκBα (p-IκBα, cat.no. 2859), IκBβ (cat.no. 8635), phospho-IκBβ (p-IκBβ, cat.no. 4921), ERK1/2 (cat.no. 4695), phospho-ERK1/2 (p-ERK1/2, cat.no. 4370), JNK (cat.no. 9252), phospho-JNK (p-JNK1/2, cat.no. 4668) and glyceraldehyde phosphate dehydrogenase (GAPDH) (cat.no. 2118) were purchased from Cell Signaling Technology (Beverly, USA). Antibodies against NIBP (cat.no. 16014-1-AP) were obtained from Proteintech Group (Rosemont, USA). PVDF membrane (cat.no. 162–0181) was obtained from Bio-Rad Laboratories (Hercules, USA). Pierce ECL Western Blotting Substrate (cat.no. 32209) was obtained from Thermo Fisher Scientific (Waltham, USA). Blasticidin S (cat.no. 203351) was purchased from EMD Millipore (Darmstadt, Germany). TNF-α (cat.no. T6674) and Matrigel (cat.no. E1270) were purchased from Sigma-Aldrich Co. (St. Louis, USA). The 24-well transwell plates (cat.no.3422) were obtained from Corning Inc. (Corning, USA).

Mouse intestine tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were then prepared and performed with immunohistochemistry staining with phospho-p65 (p-p65) (Cell Signaling Technology, Danvers, MA, USA, 93H1, 3033S) and phospho-IκB-ɑ (p-IκB-ɑ) (Cell Signaling Technology, Danvers, MA, USA, 14D4, 2859S), to analysis the phosphorylation state of phospho-p65 and -IκB-ɑ. The positive cell number was counted manually. The quantification was done by the mean value of positive cell number ratio between the treatment group and control group.