Investigating rTsSPc and RACK1 Colocalization in Caco-2 Cells

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)].