Rabbit anti β tubulin

Protein extraction and Western blot were performed as we previously reported.¹⁴ The primary antibodies were as follows: rabbit anti‐NOD2 (1: 1000, Proteintech, Wuhan, China) and rabbit anti‐β‐tubulin (1: 1000, Proteintech, Wuhan, China). The activation of the NF‐κB pathway was evaluated by NF‐κB pathway antibody kit (#9936, Cell Signaling Technology, USA). The secondary antibodies were anti‐rabbit IgG, HRP‐linked antibody (1:10,000, Wanleibio, Shenyang, China).

Total protein was extracted by using RIPA lysis buffer containing 1% phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China) after being lysed on ice, ultrasonicated and centrifuged. Based on the protein concentration determined by the BCA protein detection kit (Beyotime, China), an equally concentrated protein mixture was prepared with loading buffer solution (Biosharp, Hefei, China). The target proteins were separated via 8% or 10% SDS‒PAGE gel. Then proteins were subsequently transferred to a PVDF membrane at 300 mA for 90 min to complete the transmembrane. After 5% skim milk powder was added for 1 h, the membrane was incubated with primary antibodies at 4 ℃ overnight, which include rabbit anti-FBXO28 antibody (1:1000; abcam), rabbit anti-TGF-b1antibody (1:1000; abways), rabbit anti-Smad2/3 antibody (1:1000; CST), rabbit anti-p-Smad2/3 antibody (1:1000; CST), rabbit anti-N-cadherin antibody (1:1000; Proteintech), rabbit anti-E-cadherin antibody (1:1000; Proteintech), mouse anti-GAPDH antibody (1:5000; Proteintech) and rabbit anti-β-Tubulin (1:2000; Proteintech). After the corresponding secondary antibody was added and incubated at room temperature for 1 h, the proteins were visualized via an enhanced chemiluminescence kit (Meilunbio, China).