1. Date of download: 6/1/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Aquaporin 4 Expression and Tissue Susceptibility to Neuromyelitis Optica JAMA Neurol. 2013;70(9):1118-1125. doi:10.1001/jamaneurol.2013.3124 Aquaporin 4 (AQP4) Messenger RNA (mRNA) Expression Quantitative polymerase chain reaction analysis of AQP4 mRNA expression in 3 BL6 mice using SYBR-Green–based (Invitrogen) chemical analysis (A) and 2 human control tissue samples using SYBR-Green–based and gene expression assay (TaqMan; ABI) chemical analysis (B). Tissues analyzed were liver (L), kidney (K), muscle (M), frontal lobe (Br), brainstem (BS), hypothalamus (HyT), spinal cord (SC), and optic nerve (ON). Results are expressed as a mean ratio of the samples tested relative to a reference gene, hypoxanthine guanine phosphoribosyl transferase 1 (HPRT). In mouse tissue, M1 and M23 AQP4 isoforms were evaluated separately; in human tissue, total AQP4 (M1 and M23 isoforms) mRNA was evaluated. SYBR-Green–based and TaqMan chemical analysis methods show quantitatively different but similar relative levels of expression. Figure Legend:

2. Date of download: 6/1/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Aquaporin 4 Expression and Tissue Susceptibility to Neuromyelitis Optica JAMA Neurol. 2013;70(9):1118-1125. doi:10.1001/jamaneurol.2013.3124 Aquaporin 4 (AQP4) Protein ExpressionAnalysis of AQP4 protein expression by sodium dodecyl sulfate–18% polyacrylamide gel electrophoresis in neuromyelitis optica–relevant tissues from representative mouse (A), rat (B), and human (C) tissues. Proteins analyzed are from the cerebral hemisphere (Br), brainstem (BS), optic nerve (ON), spinal cord (SC), kidney (K), and liver (L). The M1 and M23 lanes were loaded with proteins isolated from HEK293 cells transfected separately with human M1 or M23 AQP4 isoform pcDNA. Loading was optimized to distinguish M1 from M23; differences in β-actin reflect the differences in protein loading. Densitometry results are summarized in the graphs to the right of the gels. Figure Legend:

3. Date of download: 6/1/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Aquaporin 4 Expression and Tissue Susceptibility to Neuromyelitis Optica JAMA Neurol. 2013;70(9):1118-1125. doi:10.1001/jamaneurol.2013.3124 Aquaporin 4 (AQP4) Protein Aggregation in Central Nervous System (CNS) Tissue Analysis of protein aggregation by blue native polyacrylamide gel electrophoresis in CNS tissue from a BL6 mouse (A), Lewis rat (B), and human control (C). Gels were loaded to optimize the visualization of upper-order aggregates. Proteins analyzed are from the cerebral hemisphere (Br), spinal cord (SC), and optic nerve (ON). Protein molecular weights correspond to a protein standard designed for molecular weight estimation of aggregated proteins (NativeMark Protein Standard; Invitrogen). Figure Legend:

4. Date of download: 6/1/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Aquaporin 4 Expression and Tissue Susceptibility to Neuromyelitis Optica JAMA Neurol. 2013;70(9):1118-1125. doi:10.1001/jamaneurol.2013.3124 Cerebral Hemisphere (Br) vs Optic Nerve (ON) Aquaporin 4 (AQP4) AggregationA, Blue native polyacrylamide gel electrophoresis (BN-PAGE) comparing aggregated AQP4 in ON vs Br tissue. Protein molecular weights correspond to a protein standard designed for molecular weight estimation of aggregated proteins (NativeMark Protein Standard; Invitrogen). B, Two-dimensional protein analysis wherein lanes from a 3% to 12% nondenaturing BN-PAGE gel are cut and electrophoresed in a second dimension on a denaturing sodium dodecyl sulfate–12% polyacrylamide gel (12% SDS-PAGE). The long horizontal arrow is in the direction of electrophoresis for the single-dimension 3% to 12% BN-PAGE. The vertical arrow is in the direction of electrophoresis for the second-dimension 12% SDS-PAGE. The short horizontal arrows indicate the positions of M1 and M23 isoforms of AQP4 protein. Figure Legend:

5. Date of download: 6/1/2016 Copyright © 2016 American Medical Association. All rights reserved. From: Aquaporin 4 Expression and Tissue Susceptibility to Neuromyelitis Optica JAMA Neurol. 2013;70(9):1118-1125. doi:10.1001/jamaneurol.2013.3124 Messenger RNA (mRNA), Protein Expression, and Aggregation in a Patient With Neuromyelitis Optica (NMO) Compared With ControlsComparison of aquaporin 4 (AQP4) mRNA (A), protein (B), and protein aggregation (single-dimension blue native polyacrylamide gel electrophoresis [BN-PAGE] [C] and 2-dimensional BN-PAGE [D]) between a single patient with NMO and human control samples. The optic nerve (ON) was studied using the SYBR-Green (Invitrogen) technique for mRNA expression from a single patient with NMO and from 5 control ONs (A) and from a single control (B through D) in cerebral hemisphere (Br), spinal cord (SC), ON, and kidney (K) for protein expression and aggregation. Messenger RNA expression is intermediate in NMO. Sample loading was performed to optimize the distinction of M1 from M23; β-actin serves as a comparator protein to control for differences in protein loading. Protein molecular weights (C) correspond to a protein standard designed for molecular weight estimation of aggregated proteins (NativeMark Protein Standard; Invitrogen). 12% SDS-PAGE indicates sodium dodecyl sulfate–12% polyacrylamide gel. Arrows are explained in the legend to Figure 4. Figure Legend: