Rat MuSK Antibody

Detection of Mouse MuSK by Western Blot

DOK7 gene therapy suppresses motor nerve terminal degeneration at the NMJ in ALS miceAForelimb grip strength of wild‐type (WT) mice&that of control&AAV‐D7 treatment groups of ALS mice at P84 (WT, n = 5 mice; control group, n = 5 mice; treatment group, n = 6 mice). Values are means ± SEM. **P = 0.0016 (WT vs. control group)&0.0003 (WT vs. treatment group) by one‐way analysis of variance (ANOVA) with Bonferroni's post hoc test. n.s., not significant.BThe difference in cycle threshold ( delta Ct) between the human SOD1‐G93A transgene&the reference mouse apob gene. To calculate the human transgene level, the delta Ct value of hSOD1 was subtracted from the delta Ct value of apob (control group, n = 5 mice; treatment group, n = 6 mice).C–GWT or ALS mice treated or not with 1.2 × 1012 vg of AAV‐D7 at P90 subjected to the following assays at P120. Tyrosine phosphorylation of MuSK or AChR in the hind‐limb muscle. MuSK or AChR beta 1 subunit (AChR beta 1) immunoprecipitates (IP) from whole‐tissue lysates (WTLs) of the hind‐limb muscle immunoblotted for phosphotyrosine (pY), MuSK,&AChR beta 1. WTLs blotted for Dok‐7‐EGFP, human SOD1,&GAPDH (C). Whole‐mount staining of NMJs on the diaphragm muscle. Motor nerve terminals (green)&postsynaptic AChRs (red) stained with anti‐synapsin‐1 antibodies& alpha ‐bungarotoxin, respectively. Expression of Dok‐7‐EGFP fusion protein (gray) was monitored by EGFP. Scale bars, 50 μm. NT, not treated (D). The size of AChR cluster (E), the size of motor nerve terminal (F),&innervation ratio (G) (WT‐NT, n = 5 mice; ALS‐NT, n = 5 mice; ALS‐AAV‐D7, n = 6 mice). Values are means ± SD. (E) ***P < 0.0001; (F) **P = 0.0077, ***P = 0.0001; (G) *P = 0.0134, ***P < 0.0001 by one‐way ANOVA with Dunnett's post hoc test.Source data are available online for this figure. Image collected&cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/28490573), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of MuSK by Western Blot

MuSK kinase activity fails to induce MuSK internalization. (A) Muscle cells expressing MuSK wildtype (WT), MuSK kinase-active (KA) or MuSK kinase-dead (KD) were treated with cycloheximide for the indicated time periods (in minutes). Surface proteins were isolated using biotinylation followed by streptavidin pulldown. Protein expression was determined by immunoblotting using antibodies against Lrp4, MuSK, AChR alpha and IR beta. (B) Quantification of surface protein expression in muscle cells expressing MuSK wildtype (blue), kinase-active (gray) and kinase-dead (orange) is shown over a time course of 6 h. Isolated surface proteins were normalized against IR beta. Timepoint 0 was set to 1. Data are presented as means ± SEM, n > 5. (C) Quantification of total protein expression as function of time. Total protein samples of MuSK wildtype (blue), kinase-active (gray) and kinase-dead (orange) were normalized against Actin. Timepoint 0 was set to 1. Data are presented as means ± SEM; n > 5. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35370548), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of MuSK by Western Blot

Lrp4, MuSK and AChR internalization takes place independent of Dynamin. Myotubes were pretreated with cycloheximide with or without Dyngo-4a and subsequently stimulated with 5 nM Agrin for the indicated time periods (in minutes). Surface proteins were isolated using biotinylation followed by streptavidin pulldown. (A) Samples were subjected to SDS-PAGE and analyzed by immunoblotting. Surface expression was determined using antibodies against Lrp4, MuSK, AChR alpha and IR beta. (B) Quantification of surface protein expression as function of time and Dyngo-4a treatment (− Dyngo-4a, blue; + Dyngo-4a, orange) is shown. Isolated surface proteins were normalized against IR beta. Timepoint 0 was set to 1. Data are presented as means ± SEM; n > 4. Ag, Agrin; Dg, Dyngo-4a; UT, untreated. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35370548), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of MuSK by Western Blot

Agrin stimulation increases MuSK and AChR internalization but does not affect Lrp4 internalization. Muscle cells were pretreated with cycloheximide and incubated with or without 5 nM Agrin for the indicated time periods (in minutes). Surface proteins were isolated using biotinylation followed by streptavidin pulldown. (A) Samples were subjected to SDS-PAGE and analyzed by immunoblotting. Surface and total protein expression was determined using antibodies against Lrp4, MuSK, AChR alpha, IR beta and Actin. (B) Quantification of surface protein expression as function of time and Agrin treatment (− Agrin, blue; + Agrin, orange) is shown. Isolated surface proteins were normalized against IR beta. Timepoint 0 was set to 1. Data are presented as means ± SEM; n > 9. (C) Quantification of total protein expression as function of time and Agrin treatment (− Agrin, blue; + Agrin, orange). Total protein samples were normalized against Actin. Timepoint 0 was set to 1. Data are presented as means ± SEM; n > 5. (D) Muscle cells were pretreated with cycloheximide in the presence or absence of Primaquine (Pq) followed by stimulation with 5 nM Agrin for the indicated time periods (in minutes). Surface proteins were isolated using biotinylation followed by streptavidin pulldown. Samples were subjected to SDS-PAGE and analyzed by immunoblotting. Quantification of surface protein expression as function of time and Primaquine treatment (− Primaquine, orange; + Primaquine, gray) is shown. Isolated surface proteins were normalized against IR beta. Timepoint 0 was set to 1. Data are presented as means ± SEM; n > 3. *p < 0.05; **p < 0.01; ***p < 0.001. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35370548), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of MuSK by Western Blot

MuSK endocytosis occurs independent of Lrp4. (A) Wild-type C2 cells and C2/Lrp4-KO cells were treated with cycloheximide for the indicated time points (in minutes). Biotinylation was used to isolate surface proteins followed by analysis by immunoblotting using antibodies against MuSK, AChR alpha and IR beta. (B) Quantification of MuSK and AChR surface protein expression in C2 wild-type cells (blue) and C2/Lrp4-KO cells (Exon3#2: orange; Exon3#9: gray) is shown over a time course of 6 h. Isolated surface proteins were normalized against IR beta. Timepoint 0 was set to 1. Data are presented as means ± SEM, n > 4. (C) Quantification of total protein expression as function of time in C2 wild-type cells (blue) and C2/Lrp4-KO cells (Exon3#2: orange; Exon3#9: gray). Total protein samples were normalized against Actin. Timepoint 0 was set to 1. Data are presented as means ± SEM; n > 4. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/35370548), licensed under a CC-BY license. Not internally tested by R&D Systems.