Fig 1: Immunoblots showing that IgLON5 is spontaneously shed and cleaved at the GPI-anchor. Immunoblots of different protein preparations from IgLON5-HEK probed with IgLON5 and actin antibodies: cell lysate (1); media obtained from 10 P60 plates with IgLON5-HEK cells (2); membrane preparations from one P60 plate untreated (3) and treated with PI-PLC (4); media from one P60 plate untreated (5) or treated with PI-PLC (6). Arrow indicates the glycosylated band of IgLON5 of approximately 60 kDa and the asterisk points the 37 kDa unglycosylated band. Only the lysate lane shows a beta-actin band, ruling out that the IgLON5 band detected in the other lanes is derived from dead IgLON5-HEK cells.
Fig 2: 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.
Fig 3: (A) Soluble IgLON5 is able to self-interact in trans and to bind to IgLON1-5. HEK cells, un-transfected (Unt) and transfected with IgLON1 to 5 incubated with rIgLON5-Fc protein (Top row) or rControl-Fc protein (second row). IgLON5 reactivity was detected with anti-Fc IgG-594 (Red fluorescence). The strongest IgLON5 interaction is observed with IgLON1, 3 and 4. No IgLON5 immunoreactivity was detected in untransfected HEK cells or when cells were incubated with rControl-Fc protein (bottom row). (B) Effect of IgLON5-abs on the binding of soluble IgLON5 to hippocampal neurons. IgLON5 immunofluorescence of live rat hippocampal neurons incubated with biotinylated rIgLON5-Fc (panel 1) or biotinylated rCtrl-Fc (2); Pre-incubation of biotinylated rIgLON5-Fc with CSF from patient 1 (100% IgLON5-IgG4) abolished the cell surface binding of IgLON5 (3), whereas pre-incubation with CSF from a patient with Alzheimer disease (4) or anti-NMDAR encephalitis (5) did not interfere with the binding of IgLON5 to the cell surface of neurons. Scale bar=20um.
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