Mechanism of TJs Disruption by rTsSPc

To confirm that rTsSPc binding to RACK1 damaged the TJs integrity through activating ERK1/2 MAPK pathway and down-regulating expression of TJs proteins, RACK1 inhibitor harringtonolide (HO; 40 μM; MedChemExpress, USA) and ERK1/2 pathway inhibitor PD98059 (20 μM; MCE, USA) were used in this study [61 (link),62 (link)]. Caco-2 cells were pre-treated with the two inhibitors at 37 °C for 24 h, respectively. Additionally, rabbit anti-human RACK1 antibody (1: 100; Servicebio) was also used to block the RACK1 receptor of Caco-2 cells. Then, the inhibitors-treated or antibody-blocked cells were incubated with rTsSPc (20 μg/ml) at 37 °C for 2 h. The IIFT was performed to investigate the expression and location of TJs proteins in rTsSPc-incubated Caco-2 cells [33 (link)]. Total RNAs were extracted from treated Caco-2 cells using TRIzol (Invitrogen), and expression levels of TJs mRNA in treated Caco-2 cells were ascertained by qPCR. Moreover, after the above-treated Caco-2 cells were washed using TBST, soluble cell proteins were prepared and Western blot were carried out, and rabbit antibody against occludin, claudin-1, claudin-2 and rat anti-E-cad antibody were used to detect the expression of TJs proteins in rTsSPc-incubated Caco-2 cells which were pre-treated with inhibitors or pre-blocked with antibody [21 (link)].