Figure 2. Effects of patients' antibodies on DPPX and Kv4.2 are reversible.

(A–D) Clusters of dipeptidyl-peptidase–like protein 6 (DPPX) in cultured neurons incubated for 3 days with pooled control immunoglobulin G (IgG), pooled patients’ (cases 4 and 7) IgG without recovery, and pooled patients’ (cases 4 and 7) IgG for 3 days followed by 4 or 7 days of recovery (fresh media without antibodies). Scale bars = 10 μm. Boxed dendrites are shown at higher magnification below (×100/1.3 numerical aperture oil objective). Note the reduction of clusters of DPPX caused by 3 days’ exposure to patients' antibodies and the progressive restoration of the density of DPPX clusters after neurons are allowed to recover for 4 to 7 days. (E) Graphic representation of the density of DPPX clusters (median with interquartile range) after treatment for 3 days with control or patients' IgG and after 4 to 7 days of recovery (4 independent experiments, 15 neurons per experiment in each condition). (F) An immunoblot of biotinylated surface proteins of hippocampal neurons treated with pooled control IgG and pooled patients’ IgG for 3 days without or with recovery for 4 to 7 days. The protein bands are demonstrated with commercial antibodies specific for DPPX and Kv4.2; NR1 and transferrin receptor (TfR) are used as loading controls. Note that patients' antibodies cause a substantial decrease of levels of DPPX and Kv4.2 after a 3-day incubation and that the levels of cell-surface proteins are progressively restored after 4 to 7 days. (G) Quantitative densitometry analysis of panel F; the data were normalized to the values of control IgG and are represented as median with SEM of 3 independent experiments (for each experiment, immunoblots were repeated, n = 6). All statistical analyses: Kruskal-Wallis test followed by Dunn test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.