Article Figures & Data
Figures
- Figure 1 Patients' sera recognize surface epitopes of Caspr2
HEK293 T cells were transiently transfected to express Caspr2, stained with a series of patient sera live, and then stained with a commercial antibody to Caspr2 after permeabilization and fixation. Nuclei of transfected and untransfected cells were stained with DAPI (blue). Surface epitopes of Caspr2 were recognized by all 10 patients' sera (4 representative samples [S1–S4] are shown). Scale bar = 10 μm.
- Figure 2 Caspr2 recognition does not depend on glycosylation or tertiary structure
(A) Cells were transfected to express Caspr2 in the presence of tunicamycin or vehicle (DMSO). Tunicamycin treatment resulted in intracellular localization of Caspr2, but the transfected cells were still recognized by patients' sera and a commercial antibody to Caspr2 (ab33994). Scale bar = 10 μm. (B) Lysates from cells transfected with empty vector, cells transfected with Caspr2 treated with vehicle (DMSO), Caspr2 cells treated with tunicamycin (Tun), and Caspr2 lysates treated with PNGase F were probed on Western blot with a commercial Caspr2 antibody and patient sera.
- Figure 3 Single-domain deletion constructs were recognized by patients' sera
(A) Single-domain deletion constructs were generated as shown. (B) Each of these constructs was recognized by patients' sera on Western blot probed with a commercial antibody to Caspr2 (Caspr2) and patients' sera (sera 1).
- Figure 4 Some single-domain deletion constructs showed decreased labeling by patients' sera
(A) Cultured cells were transfected to express wild-type and single-domain deletion constructs. All constructs were expressed on the cell membrane and all were recognized by a panel of patients' sera. Scale bar = 10 μm. (B) The relative staining in the human and commercial channels was quantified for the various deletion constructs. Only ΔDisc differed significantly from full-length Caspr2 using this method.
- Figure 5 The Disc domain contains a target epitope
(A) A construct (Δ182-Lam4) with all extracellular subdomains except the Disc domain was created. (B) This construct was recognized by 9 of 10 sera in transfected cells. Scale bar = 10 μm. (C) Western blot of lysates from cells transfected with an empty vector, wild-type Caspr2, or the Δ182-Lam4 construct were probed with human sera and then reprobed with a rabbit antibody to Caspr2. The Δ182-Lam4 protein is significantly smaller than wild-type Caspr2 but is still recognized by both human and rabbit antibodies.
- Figure 6 Multiple target epitopes exist in the N-terminal domains of Caspr2
(A) Multidomain deletion constructs of Caspr2 were generated to determine which domains contain target epitopes. (B) These constructs were transfected into cultured cells and immunostained with patient sera and a rabbit antibody to Caspr2 (ab33994). Scale bar = 10 μm. (C) The results for 7 patient sera are shown. See text for discussion.
Additional Files
Data Supplement
Files in this Data Supplement:
- Table e-1 - Microsoft Word file
- Figure e-1 - .jpg file
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