Rat MuSK Antibody

Acetylcholine Receptor Clustering Mediated by MuSK and Neutralization by Rat MuSK Antibody.

Recombinant Rat Agrin (Catalog # 550-AG) induces MuSK-dependent acetylcholine receptor clustering on myotubes differentiated from the C2C12 mouse myoblast cell line in a dose-dependent manner (orange line). Acetylcholine Receptor Clustering elicited by Recombinant Rat Agrin (16 ng/mL) is neutralized (green line) by increasing concentrations of Goat Anti-Rat MuSK Antigen Affinity-purified Polyclonal Antibody (Catalog # AF562). The ND₅₀ is typically 0.5-2 µg/mL.

Detection of Human MuSK by Immunocytochemistry/ Immunofluorescence

MuSK patient IgG4 or IgG1-3 do not induce endocytosis of MuSK.Patient plasma or purified IgG1-3 or IgG4 were applied at 0.17nM final concentration of MuSK antibody to HEK293 cells expressing MuSK at the cell surface. Cells were either incubated at 4°C to prevent, or at 37°C to allow, endocytosis. Patient antibody binding was visualised by addition of a secondary fluorescent anti-human antibody at the end of the experiment. (A) An example of cells that were incubated with IgG1-3 from patient 9. Robust staining was observed after 6 hours incubation at both temperatures. Scale bar=25µm. (B) Staining was scored by two individuals as described in methods, and the scores were normalised to the score at 0 hours for each condition. A slight decrease in staining was observed for cells incubated at 37°C, and pre-fixed cells also showed this reduction. (C) As a positive control for endocytosis, IgG1-3 or IgG4 was cross-linked by addition of Alexa Fluor 568-conjugated anti-human IgG prior to incubation. Example images show cells treated with patient 9 IgG4 and IgG1-3 in the presence and absence of cross-linking anti-human IgG after 6 hours incubation at 37°C. There is a clear difference when the cross-linking secondary antibody is present. Scale bar =50µm (D) Cross-linking by the secondary antibody induced internalisation of human anti-MuSK antibodies as well as MuSK-EGFP as early as 30 minutes, as observed by confocal microscopy. An example image of a cell from a confocal z-stack with orthogonal side-views is shown. The arrow shows internalised secondary Alexa Fluor 568-conjugated anti-human IgG (red) colocalised with EGFP-tagged MuSK (green). Scale bar =5µm. Image collected and cropped by CiteAb from the following open publication (https://dx.plos.org/10.1371/journal.pone.0080695), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human MuSK by Immunocytochemistry/ Immunofluorescence

MuSK patient IgG4 or IgG1-3 do not induce endocytosis of MuSK.Patient plasma or purified IgG1-3 or IgG4 were applied at 0.17nM final concentration of MuSK antibody to HEK293 cells expressing MuSK at the cell surface. Cells were either incubated at 4°C to prevent, or at 37°C to allow, endocytosis. Patient antibody binding was visualised by addition of a secondary fluorescent anti-human antibody at the end of the experiment. (A) An example of cells that were incubated with IgG1-3 from patient 9. Robust staining was observed after 6 hours incubation at both temperatures. Scale bar=25µm. (B) Staining was scored by two individuals as described in methods, and the scores were normalised to the score at 0 hours for each condition. A slight decrease in staining was observed for cells incubated at 37°C, and pre-fixed cells also showed this reduction. (C) As a positive control for endocytosis, IgG1-3 or IgG4 was cross-linked by addition of Alexa Fluor 568-conjugated anti-human IgG prior to incubation. Example images show cells treated with patient 9 IgG4 and IgG1-3 in the presence and absence of cross-linking anti-human IgG after 6 hours incubation at 37°C. There is a clear difference when the cross-linking secondary antibody is present. Scale bar =50µm (D) Cross-linking by the secondary antibody induced internalisation of human anti-MuSK antibodies as well as MuSK-EGFP as early as 30 minutes, as observed by confocal microscopy. An example image of a cell from a confocal z-stack with orthogonal side-views is shown. The arrow shows internalised secondary Alexa Fluor 568-conjugated anti-human IgG (red) colocalised with EGFP-tagged MuSK (green). Scale bar =5µm. Image collected and cropped by CiteAb from the following open publication (https://dx.plos.org/10.1371/journal.pone.0080695), licensed under a CC-BY license. Not internally tested by R&D Systems.