Fig 1: Analysis of IgLON5 antibody subclasses. a Example of anti-IgLON5 IgG subclasses in three different sera. HEK293 cells were transfected with IgLON5 and incubated with three sera with IgLON5 antibodies followed by antibodies against total IgG or specific IgG subclasses. Positive immunoreactivity is shown in green. Nuclei counterstained with DAPI (blue). Scale bar = 10 µm. b IgG subclass percentages of IgLON5 antibodies in the 15 positive serum samples analyzed by flow cytometry
Fig 2: IgG1 IgLON5 antibodies internalize IgLON5 clusters. Hippocampal neurons treated for 3 days with total IgG, IgG1, and IgG4 IgLON5 antibodies. The immunofluorescence strategy to differentiate surface and internal human IgG bound to IgLON5 is conducted as in Fig. 8. The IgG1 antibodies alone could reproduce the same effects seen with the total IgG; meanwhile, the IgG4 did not produce internalization. Scale bar = 5 µm
Fig 3: IgLON5 antibodies irreversible decrease IgLON5 clusters on cell surface. a Immunofluorescence on hippocampal neurons treated for 7 days (14DIV to 21DIV) with control or patient with anti-IgLON5 IgG. The surface clusters are drastically reduced by the anti-IgLON5 IgG, and the effect is irreversible because 7 days after removing the anti-IgLON5 IgG the reduced number of surface IgLON5 clusters persisted. Scale bar = 5 µm. b Quantification of the decrease of IgLON5 clusters on the dendrite surface in a time-course treatment of 1, 3, and 7 days, and 7 days after removing the anti-IgLON5 IgG. ***p < 0.005, ****p < 0.0001. c Synaptic markers (PSD95, synapsin-I) were not affected by the anti-IgLON5 IgG treatment
Fig 4: Comparison of the time-dependent decrease of neuronal cell surface IgLON5 clusters after treatment with total IgG of two patients with different levels of anti-IgLON5 IgG1 antibodies. On days 1 and 3, treatment with IgG of the patient with the lower level of IgG1 IgLON5 antibodies (7 % of total IgLON5 antibodies) produced a similar decrease of IgLON5 clusters as the IgG of the patient with average level of IgG1 antibodies (23 % of total IgLON5 antibodies). (**p = 0.001, ****p < 0.0001, comparing with control IgG, one-way ANOVA with Bonferroni’s multiple comparison test). In contrast, on day 7, the sample of the patient with lower levels of IgG1 antibodies did not decrease further the levels of IgLON5, whereas the sample of the patient with average level of IgG1 IgLON5 antibodies produced an additional decrease of IgLON5 clusters (****p < 0.0001) that was statistically significant comparing with day 3 (*p = 0.03)
Fig 5: Immunoblots of co-tranfected HEK cells immunoprecipitated with patients’ IgLON5 antibodies. HEK cells co-transfected with IgLON5 and each of the other IgLON family members: first row (IgLON5 + IgLON1), 2nd row IgLON2 (5+2), 3rd row (5+3), and 4th row (5+4). The 5th row corresponds to HEK cells transfected only with IgLON5, and the 6th row HEK cells co-transfected with IgLON5 and CASPR2 (5+C). Left column: Whole cell lysates (WCL); middle column: Immunoprecipitates of transfected HEK cells using patients’ IgLON5-abs (IP+); right column: Immunoprecipitates of transfected cells using Ctrl-abs (IP). Each row was incubated with commercial antibodies against the corresponding proteins: IgLON1, 2,3,4,5 and CASPR2.
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