Rabbit anti human rack1 antibody

To investigate co-localization of rTsSPc and receptor for activated protein C kinase 1 (RACK1) in Caco-2 cells, the IIFT was performed as reported before [47 (link),48 (link)]. Briefly, confluent Caco-2 monolayers were apically incubated with 20 μg/ml of rTsSPc for 2 h at 37 °C. IIL ES antigens were used as a positive control and PBS as a negative control. After interaction, the cells were washed three times with washing buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 6.8) to eliminate unbound protein molecules. The cells were fixed in 4% paraformaldehyde at room temperature for 15 min. To reduce non specificity, cells were blocked for 2 h with 1% bovine serum albumin (BSA; Sigma). Then, the cells were probed overnight at 4 °C by rabbit anti-human RACK1 antibody (1:1 000; Servicebio, Wuhan, China) and mouse anti-rTsSPc immune serum (1:100). Then, the cells were stained by using Alexa Fluor 488 labeled goat anti-mouse IgG (1:100; Abways, Shanghai, China) and CY3 labeled goat anti-rabbit IgG (1:100; Servicebio) as the secondary antibody. After being washed with PBST, a 4′,6-diamidino-2-phenylindole (DAPI) was used for fluorescence staining of the cell nucleus. Finally, the cells were observed under a laser confocal fluorescence microscopy [24 (link),42 (link),49 (link)].

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)].