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. 2017 Jul 19;7:326. doi: 10.3389/fcimb.2017.00326

Figure 1.

Figure 1

Identification of IPEC-J2 receptor for Salmonella Choleraesuis FimH adhesion. (A) Scheme of the receptor identification procedures. (B) 2D electrophoresis of the IPEC-J2 cell lysate. IPEC-J2 cells were lysed in extraction buffer and stained with Coomassie brilliant blue. The horizontal axis shows the isoelectric point, and the vertical axis shows molecular weight (kDa). The molecular mass range of proteins cut from the gel is marked by black lines. (C) Far-Western blotting analysis of CFimH binding to proteins from the IPEC-J2 cell lysate. Cell lysates, equivalent to 300 μg of protein, were separated in two dimensions (pH and molecular weight) and transferred onto nitrocellulose. The horizontal axis shows the isoelectric point, and the vertical axis shows molecular weight (kDa). (D) Coomassie-stained 1D electrophoresis of IPEC-J2 glycoprotein fraction (20 μg of protein per line) obtained by ConA affinity chromatography. The migration of protein standards (in kDa) is indicated on the left. (E) Far-Western blotting analysis of CFimH binding to the ConA fraction of the IPEC-J2 cell line. Cell lysates, equivalent to 20 μg of protein, were separated by SDS–PAGE and transferred onto nitrocellulose. The migration of protein standards (in kDa) is indicated on the left. (F) Detection of calreticulin by Western blotting with anti-calreticulin rabbit monoclonal antibodies in IPEC-J2 whole cell lysate and IPEC-J2 membrane fraction. Cell lysates, equivalent to 20 μg of protein, were separated by SDS–PAGE and transferred onto nitrocellulose. (G) Flow cytometry analysis of IPEC-J2 cells. The cells were detached with Accutase, washed with PBS, incubated with rabbit anti-calreticulin antibody followed by PE-conjugated secondary antibody, fixed with 1% buffered formaldehyde and immediately analyzed on a cytometer. The percentage of CRT-positive cells is presented on the image. (H) Adherence of SCΔfimH/C to IPEC-J2 cells preincubated for 1 h with PBS buffer (control) or anti-calreticulin monoclonal antibody (Ab). Data are means ± SD in triplicate assays, which were independently repeated three times. Here and in all other assays *p < 0.05, **p < 0.01, ***p < 0.001.