Journal: Neurology® Neuroimmunology & Neuroinflammation

Article Title: Distinct Epitopes Are Associated With Clinical Phenotypes in Autoimmune Nodopathies With Anti-Contactin1 Autoantibodies

doi: 10.1212/NXI.0000000000200507

Figure Lengend Snippet: (A) Transfected HEK293 cells expressing full-length CNTN1 wild-type and deletion mutants of the extracellular Ig domains 1–6 (deletion of Ig1 domain corresponds to CNTN1ΔIg1; deletion of Ig domains 1 and 2 CNTN1ΔIg1,2; and further deletions CNTN1ΔIg1-3, CNTN1ΔIg1-4, CNTN1ΔIg1-5, and CNTN1ΔIg1-6; red). CNTN1 wild-type and deletion mutants were fused to GFP (cyan). (B) Representative Western blot of CNTN1 wild-type and deletion variants. Note, the decrease in molecular weight on increase of the deletion (white open arrowheads between 125 and 80 kDa). # marks the lower protein bands representing most likely the nonglycosylated isoforms. β-Actin was used as housekeeping protein (black arrowhead; 48 kDa). Dotted line marks cut of the nitrocellulose. (C) Quantification of the whole-cell protein fractions of CNTN1 variants normalized to β-actin after a biotinylation assay (n = 5); significance value ** p < 0.01. (D) Quantification of the surface protein fractions of CNTN1 variants normalized to pan-cadherin (pan-cad, membrane marker) (n = 3). Dotted lines in C and D point to the expression level of CNTN1 wild-type mutant. Error bars represent SEM.

Article Snippet: Samples were then transferred onto a nitrocellulose membrane (GE Healthcare, Little Chalfont, UK), followed by blocking for 1 hour with 5% milk powder (for CNTN1) or 5% BSA (for pan-cad and β-actin) in TBS-T (TBS with 1% v/v Tween 20); membranes were incubated overnight at 4°C with patient or control serum (1:250 or 1:500) or primary antibodies [anti-CNTN1 (R&D Systems, Minneapolis, MN, AF904, 1:400 or 1:500), pan-cadherin (Cell Signaling Technology, Danvers, MA, 4068S, 1:1,000), or β-actin (GeneTex - Biozol, Eching, Germany, GTX26276, 1:5,000)].

Techniques: Transfection, Expressing, Western Blot, Molecular Weight, Cell Surface Biotinylation Assay, Membrane, Marker, Mutagenesis