(A) Comparable Rapsn protein level between WT and N88K mt mice. Tissue lysates from WT, N88K mt, -/- (Rapsn null mt) diaphragms were subjected to western blotting with anti-Rapsn, and anti-α-AChR …
Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 1E–J.
(A) Generation of N88K mt mice by CRISPR-Cas9. Top, the Rapsn gene structure. Genomic sequence flanking the Asn 88 was shown below. Guidance RNA sequence, GCT CGA AAG CTA CCT GAA CC, was labeled by …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 1—figure supplement 1C, E and H.
(A, B) Low magnification NMJ EM images of WT and N88K mt, including synaptic vesicles (SVs), synaptic cleft, synaptic basal lamina (SBL), and postsynaptic junctional folds (JF). M, muscle; N, nerve; …
Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 2E, F, G, H, M and N.
(A) Comparable resting membrane potentials among WT, N88K/+, N88K mt, and N88K/- mt. P0 hemi-diaphragms at ventral, left were recorded. (B) Percentage of muscle fibers with mEPPs in 3 min of recordin…
Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figures 3A, B, D and E.
(A) Impaired ability of N88K mt Rapsn to induce AChR clustering in HEK293T cells. HEK293T cells were transfected with AChR subunits (α, β, γ, δ), along with EGFP empty vector, Rapsn-EGFP, or …
Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 4B and F.
(A) Comparable degradation rate between WT and N88K mt Rapsn in HEK293T cells. HEK293T cells were transfected with EGFP tagged WT or N88K mt Rapsn. After 24 hr, cells were separated into 6-well …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 4—figure supplement 1B.
(A) Verification of N88K mutation by genomic DNA sequencing. Genomic DNA of WT and N88K mt C2C12 was sequenced and aligned. Dashed rectangle to indicate N88K mutation. (B) Comparable Rapsn mRNA …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 4—figure supplement 2B and D.
(A) Schematic diagram of extraction of surface AChRs from C2C12 myotubes. Live C2C12 myotubes were incubated with biotin-α-BTX at 4°C for 1 hr to capture AChR complex, and then were lysed. Resulting …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 5D, F and H.
(A, B) HEK293T cells were cotransfected with HA tagged WT or N88K mt Rapsn and Flag tagged α-AChR (A) or δ-AChR (B). After 48 hr, cells were lysed and the interaction of Rapsn with subunits of AChR …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 5—figure supplement 1D.
(A) Agrin treatment induced tyrosine phosphorylation of Rapsn in cultured myotubes. WT cultured myotubes were treated with Agrin for indicated times. Cells were lysed and were incubated with …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 6D, G and I.
(A) Reduced E3 ligase activity of Rapsn by Y86F mutation in transfected HEK293T cells. (B) Quantitative data of neddylated δ-AChR in (A) (mean ± SEM), ***, p<0.001, One-way ANOVA, n = 3. (C) Reduced …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 7B, D, F, H, J, L and N.
(A) Generation of Y86F mt C2C12 cells by CRISPR-Cas9. Top, the Rapsn gene structure. Genomic sequence flanking the Y86 was shown below. Guidance RNA targeting sequence, CAT TGC TGC GCG CCA GGT TC, …
Raw data, sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 7—figure supplement 1C and E.
(A) Thigh muscles of N88K mt were infected with AAV-WT-Rapsn-EGFP (N88K + AAV-WT-EGFP), or infected with AAV-Y86F-Rapsn-EGFP (N88K + AAV-Y86F-EGFP) at E13. The resulting P0 muscles were stained with …
Sample size (n), mean, SEM, p value, statistical methods and results are presented in Figure 8B, C and D.
(A) Schematic diagram of generation of AAV vector expressing Rapsn. (B) HEK293T cells were infected with AAV-Rapsn-EGFP (AAV-WT-EGFP) or AAV-Y86F-Rapsn-EGFP (AAV-Y86F-EGFP). After 24 hr, Rapsn-EGFP …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Genetic reagent (M. musculus) | Rapsn-/- | PMID: 7675108 | ||
Genetic reagent (M. musculus) | N88K | This paper | See detail information in Materials and methods . | |
Cell line (Homo sapiens) | HEK293T | ATCC | Cat#:CRL-3216 RRID: CVCL_0042 | From ATCC; Cell identity has been confirmed by STR profiling and cell line was found to be free of Mycoplasma. |
Cell line (M. musculus) | C2C12 | ATCC | Cat#: CRL-1772 RRID: CVCL_0188 | From ATCC; Cell identity has been confirmed by STR profiling and cell line was found to be free of Mycoplasma. |
Cell line (M. musculus) | N88K mt C2C12 | This paper | See detail information in Material and methods). | |
Cell line (M. musculus) | Y86F mt C2C12 | This paper | See detail information in Material and methods. | |
Cell line (M. musculus) | Rapsn-/- C2C12, clone 11–7 | PMID: 10414969 | ||
Antibody | Mouse monoclonal anti-HA Agarose | Thermo Fisher Scientific | Cat#: 26181, RRID: AB_2537081 | 1: 40 for IP |
Antibody | Goat anti-rabbit IgG conjugated with Alexa Fluor 488 | Thermo Fisher Scientific | Cat#: A-11008 RRID: AB_10563748 | IHC (1:500) |
Antibody | Rabbit polyclonal anti-Actin | Cell Signaling Technology | Cat #: 4967, RRID: AB_330288 | WB (1:10000) |
Antibody | Rabbit polyclonal anti- α-Actin | Abcam | Cat #: ab52218 RRID: AB_870573 | WB (1:1000) |
Antibody | Rabbit polyclonal anti-Flag | Sigma-Aldrich | Cat #: F7425 RRID: AB_439687 | WB (1:1000) |
Antibody | Mouse monoclonal anti-GAPDH (Clone, 6C5) | Santa Cruz Biotechnology | Cat #: sc-32233, RRID: AB_627679 | WB (1:10000) |
Antibody | Rabbit polyclonal anti-neurofilament | Cell Signaling Technology | Cat#: 2837, RRID: AB_823575 | IHC (1:1000) |
Antibody | Rabbit polyclonal anti-synapsin | Cell Signaling Technology | Cat#: 5297, RRID: AB_2616578 | IHC (1:1000) |
Antibody | Mouse monoclonal anti-Ubiquitin (Clone, Ubi-1) | Abcam | Cat#: ab7254, RRID: AB_305802 | WB (1:1000) |
Antibody | Rabbit polyclonal anti-GFP | Cell Signaling Technology | Cat#: 2555, RRID: AB_10692764 | WB (1:1000) |
Antibody | Mouse monoclonal P-Tyr-100 | Cell Signaling Technology | Cat #: 9411 RRID: AB_331228 | WB (1:1000) |
Antibody | Mouse monoclonal Anti-Flag affinity gel (Clone, M2) | Sigma-Aldrich | Cat#: A2220, RRID: AB_10063035 | 1: 50 for IP |
Antibody | Rabbit polyclonal anti-HA | Sigma-Aldrich | Cat#: H6908, RRID: AB_260070 | WB (1:2000) |
Antibody | Mouse monoclonal anti-Rapsn (Clone 1234) | Abcam | Cat#: ab11423, RRID: AB_298028 | WB (1:1000) |
Antibody | Rabbit polyclonal anti-Transferrin | Abcam | Cat#: ab82411, RRID: AB_1659060 | WB (1:1000) |
Antibody | Mouse monoclonal anti-δ-AChR (Clone, 88B) | Thermo Fisher Scientific | Cat#: MA3-043, RRID: AB_2081037 | WB (1:1000) |
Antibody | Rabbit polyclonal anti-Rapsn | PMID: 18940591 | WB (1:1000) | |
Antibody | Goat polyclonal anti-α-AChR | PMID: 3484485 | WB (1:1000) | |
Antibody | Rabbit polyclonal anti-β-AChR | PMID: 3484485 | WB (1:1000) | |
Antibody | Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG | Thermo Fisher Scientific | Cat#: 32260, RRID: AB_1965959 | WB (1:5000) |
Antibody | Horseradish peroxidase (HRP)-conjugated goat anti-Mouse IgG | Thermo Fisher Scientific | Cat#: 32230, RRID: AB_1965958 | WB (1:5000) |
Antibody | Horseradish peroxidase (HRP)-conjugated goat anti-Rat IgG | Thermo Fisher Scientific | Cat#: 31470, RRID: AB_228356 | WB (1:5000) |
Recombinant Protein | Agrin | R and D Systems | Cat#: 550-AG-100 | 50 ng / ml for induction of AChR clusters in culture myotubes |
Other | Immunofluorescence of NMJ in diaphragm and muscles | PMID: 18278041 PMID: 22794264 | ||
Other | Electron microscopic analysis | |||
Other | Generation of Gene-modified C2C12 cells | PMID: 27839998 | ||
Other | Isolation of cell surface protein | PMID: 22157653 | ||
Other | Isolation of surface AChR and their associated proteins | PMID: 18940591 | ||
Other | AAV Virus production | PMID: 30626963 and Information in Addgene: https://www.addgene.org/protocols/aav-production-hek293-cells/ |