1. Date of download: 7/8/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Presence and Pathogenic Relevance of Antibodies to Clustered Acetylcholine Receptor in Ocular and Generalized Myasthenia Gravis Arch Neurol. 2012;69(8):994-1001. doi:10.1001/archneurol.2012.437 Figure 1. Cell-based assay using clustered acetylcholine receptors (AChRs) in myasthenia gravis (MG). A, Immunocytofluorescence of -AChR–, γ-AChR–, and muscle-specific kinase (MuSK)–transfected human embryonic kidney cells showing the presence of low- affinity AChR antibodies (Abs) in previously “seronegative” (SN) patients with ocular MG (OMG). The patient with MuSK-Abs (OMG2) detected using the cell-based assay also had AChR-Abs (original magnification, x1000). EGFP indicates enhanced green fluorescence protein. B, The cell-based assay scores for the OMG and generalized MG (GMG) groups of SN patients were similar (unpaired t test, P =.43). C, Quantification by fluorescence-activated cell sorting of a representative patient (SN-OMG14). The background fluorescence is assessed using nonstained cells and the regions gated. Cells in the M1 region are considered to have background fluorescence, and those in the M2 region are thought to have specific immunoglobulin G (IgG) binding. D, Comparison of flow cytometry quantification of IgG binding in SN and AChR-positive OMG. There was a significant difference in IgG binding by the serum of SN and AChR-positive patients compared with that of controls (analysis of variance with Bonferroni multiple comparisons). Figure Legend:

2. Date of download: 7/8/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Presence and Pathogenic Relevance of Antibodies to Clustered Acetylcholine Receptor in Ocular and Generalized Myasthenia Gravis Arch Neurol. 2012;69(8):994-1001. doi:10.1001/archneurol.2012.437 Figure 2. Complement activation in vitro by seronegative (SN) myasthenia gravis (MG) serum samples. Surface staining for activated C3b or membrane attack complex (MAC) (red) on human embryonic kidney cells transfected with acetylcholine receptor (AChR) and rapsyn (green) sensitized using MG serum samples. EGFP indicates enhanced green fluorescence protein. Figure Legend:

3. Date of download: 7/8/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Presence and Pathogenic Relevance of Antibodies to Clustered Acetylcholine Receptor in Ocular and Generalized Myasthenia Gravis Arch Neurol. 2012;69(8):994-1001. doi:10.1001/archneurol.2012.437 Figure 3. Neurophysiologic and clinical correlations with antibody levels. A, The orbicularis oculi (OO) single-fiber electromyographic jitter (mean consecutive difference [MCD]) values were not significantly different among the predominantly ocular, limb, or bulbar phenotypes. Error bars represent SEM. B, Correlation of the antibody against acetylcholine receptor (AChR-Ab) titers obtained from immunoprecipitation assay and the MCD of OO muscle. C, Correlation of the cell-based assay (immunoglobulin G [IgG]) scores and the MCD of OO muscle. D, Highly significant correlation of the neuromuscular jitter (MCD) values with the C3b binding scores. The dotted lines represent the upper limit of normal for AChR-Abs and mean jitter MCD in healthy control subjects. Figure Legend:

4. Date of download: 7/8/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Presence and Pathogenic Relevance of Antibodies to Clustered Acetylcholine Receptor in Ocular and Generalized Myasthenia Gravis Arch Neurol. 2012;69(8):994-1001. doi:10.1001/archneurol.2012.437 Figure 4. Electrophysiologic changes in high-dose (250-mg) immunoglobulin G (IgG) passive transfer to CD59 −/− /Daf1 −/− mice. A, Reduction in mean miniature end plate potential (MEPP) amplitudes compared with controls in both seronegative (SN) myasthenia gravis (MG) test groups (SNMG1 and SNMG2) and the positive control group (acetylcholine receptor [AChR]–MG). There were no differences in the MEPP frequencies (B), EPP (end plate potential) amplitudes (C), or quantal contents (D) in the SNMG1, SNMG2, and AChR-MG IgG–injected animals compared with the control IgG–injected group. The Kruskal-Wallis analysis of variance test was used for statistical significance, and the Dunn posttest was used to compare the difference between groups. Error bars represent SEM. Figure Legend:

5. Date of download: 7/8/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Presence and Pathogenic Relevance of Antibodies to Clustered Acetylcholine Receptor in Ocular and Generalized Myasthenia Gravis Arch Neurol. 2012;69(8):994-1001. doi:10.1001/archneurol.2012.437 Figure 5. Complement deposition at postsynaptic membrane and changes in end plate areas in passive transfer of myasthenia gravis (MG). Complement deposition was visualized using C3b binding by green fluorescent–tagged anti-human C3b and was co- localized to the end plates using red fluorescent–tagged bungarotoxin (BuTx). A, Strong complement deposition was seen in mice injected with acetylcholine receptor (AChR)–MG immunoglobulin G (IgG), with lesser staining seen with seronegative (SN) MG IgG passive transfer, where it was mainly limited to the end plates. B, There was significant reduction in the mean end plate areas in mice injected with the SNMG and AChR-MG patients' IgG. Error bars represent SEM; asterisks, P <.001. Figure Legend: