SUPPLEMENTAL INFORMATION
Generation and validation of antibodies to CdtB
To determine the role of CdtB in the development of IBS, two antibodies to CdtB from C. jejuni
81-176 were used. For the first of these (haAB), rabbits were immunized with a highly antigenic
18 amino acid peptide from C. jejuni CdtB identified through protein modeling (AnaSpec, San
Jose, CA) and the resulting anti-sera were affinity-purified (AnaSpec). For the second of these
(wAb, developed by Dr. Patricia Guerry (Naval Medical Research Center, Silver Spring, MD)), rabbits were immunized with a near-full length CdtB protein and the resulting anti-sera were used in our experiments. The antibodies were first used to probe Western blots of both recombinant CdtB protein, and of a lysate from C . jejuni 81-176. Both antibodies identified the recombinant CdtB protein (Supplemental Figure 1a,b), and also identified a band at approximately 29 kDa in the C . jejuni lysate (Supplemental Figure 1d,e), which is the molecular weight of CdtB. This band was not identified by rabbit preimmune serum, and was eliminated when the haAb antibody was blocked via preincubation with the peptide (Supplemental Figure
1c,f).
The wAb was used for the remainder of our experiments.

(a) wAb
(b) haAb
(c) haAb + haPeptide
CdtB protein
(d) wAb
(e) haAb
(f) haAb + haPeptide
C. jejuni lysate
Supplemental Figure 1. Detection of CdtB by antibodies generated to the CdtB protein and a CdtB peptide. Western blots of the purified CdtB protein (A-C) and of C.
jejuni lysate (D-E) probed with an antibody generated to near-full length CdtB (wAb) (A,D), an antibody generated to a highly antigenic CdtB peptide (hAb) (B,E), or with hAb following blocking with the hAb peptide (C,F).

Pre-immune serum
(a) (c)
Anti-CdtB
(wAb)
(b) (d)
C. jejuni-infected rats Controls
Supplemental Figure 2. Antibodies to CdtB stain endogenous neural elements of the gut in the rat ileum. Immunohistochemical staining of rat ileal tissue sections with CdtB antiserum
(wAb) (B,D) or pre-immune serum (A,C) in C.
jejuniinfected rats 2 days post gavage (A,B) or in control rats (C,D).

(a)
Pre-immune serum
(b)
Anti-CdtB (wAb)
Supplemental Figure 3. Antibodies to CdtB stain endogenous neural elements of the gut in the human ileum.
Immunohistochemical staining of human ileal tissue sections from subjects who did not have IBS with pre-immune serum (A) or CdtB antiserum (wAb) (B).

Control
IP with
CdtB
150
120
100
70
50
30
117 kDa
(Vinculin)
25
20
10
Supplemental Figure 4. Detection of vinculin in proteins immunoprecipitated from murine enteric undifferentiated neuronal cells using an anti-CdtB antibody. SYPRO Ruby staining of proteins immunoprecipitated from the cytosolic fraction of a lysate of murine enteric undifferentiated neuronal cells using an antibody to CdtB (wAb) vs. controls. The band at 117 kDa was subsequently identified as vinculin.