mouse anti ncx3  (Novus Biologicals)

Journal:

Article Title: Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

doi: 10.1172/JCI200418688

Figure Lengend Snippet: Targeted disruption of the Ncx3 gene. (a) Structure of the WT and the targeted alleles. The second exon (shaded boxes), the neomycin resistance cassette (black box), the probes used in DNA and RNA hybridization analysis (black bars underneath the second exon), as well as the DNA fragments generated after digestion with KnpI + SpeI or KpnI alone are represented. K, KpnI; H, HindIII; S, SpeI; E, EcoRI; B, BamHI. (b and c) DNA hybridization analysis of KpnI + SpeI– (b) and KpnI- (c) digested genomic DNA isolated from ES clones using the depicted probe. (d) RNA hybridization analysis. Messenger RNA (0.5 μg/lane) was hybridized with Ncx3 or GAPDH RNA probes. (e) RT-PCR analysis. Ncx3 amplicon (∼600 bp) and housekeeping gene HPRT amplicon (∼250 bp) were simultaneously amplified from gastrocnemius muscle messenger RNA by one-step RT-PCR. (f, g, and h) Western blot analysis. (f) Membrane fractions (50 μg/lane) from gastrocnemius muscle were analyzed with anti-NCX3–specific antibody. (g) Immunodetection of membrane fractions from Ncx3+/+ (lanes 1–3) and Ncx3–/– (lanes 4–6) gastrocnemius muscle analyzed with anti-NCX1– and anti-PMCA1–specific antibodies. Amount of protein loaded: lanes 1 and 4, 50 μg; lanes 2 and 5, 15 μg; lanes 3 and 6, 5 μg. (h) Membrane fractions (20 μg/lane) from FDB muscle were analyzed with anti-NCX1–specific antibody. Protein loading in (f–h) was determined by using a mouse anti-desmin monoclonal antibody.

Article Snippet: Primary antibodies. (i) Anti-mouse NCX3 exchanger; peptide DGNFTPLEGKEVDES from the mouse NCX3 protein sequence was used for rabbit immunization (Eurogentec, Herstal, Belgium).

Techniques: Hybridization, Generated, DNA Hybridization, Isolation, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Western Blot, Immunodetection

Journal:

Article Title: Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

doi: 10.1172/JCI200418688

Figure Lengend Snippet: NCX activity in FDB muscle. [Ca2+]i was measured in FDB fibers loaded with Fura-PE3. (a) Response to extracellular Na+ removal (replaced by NMDG). Graphs are representative of [Ca2+]i transients from Ncx3+/+ (n = 7) and Ncx3–/– (n = 9) FDB fibers. (b) Response to caffeine and TBQ in the presence (Krebs) or in the absence of extracellular Na+. Graphs are representative of five experiments with Ncx3+/+ FDB fibers. (c) Response to caffeine and TBQ in Ncx3–/– fibers in the absence of extracellular Na+. The graph is representative of five experiments. (d) Comparison of the Na/Ca exchange activity in response to caffeine and TBQ in Ncx3+/+ (n = 7) and Ncx3–/– (n = 7) FDB fibers. Results are displayed as the mean (bold traces) ± SEM (thin traces).

Article Snippet: Primary antibodies. (i) Anti-mouse NCX3 exchanger; peptide DGNFTPLEGKEVDES from the mouse NCX3 protein sequence was used for rabbit immunization (Eurogentec, Herstal, Belgium).

Techniques: Activity Assay

Journal:

Article Title: Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

doi: 10.1172/JCI200418688

Figure Lengend Snippet: Histological alterations in Ncx3–/– muscles. H&E-stained sections from 3-month-old Ncx3+/+ (a) and Ncx3–/– (b) gastrocnemius muscle: focus of fiber necrosis and cellular infiltrate in Ncx3–/– mice. Scale bar: 50 μm. Localization of EBD in cryosections of gastrocnemius muscles: 3-month-old EBD-injected Ncx3+/+ (c) and Ncx3–/– (d) mice were examined after 12–24 hours. Red stained structures are nuclei, green stained areas are cytosol, and blue stained areas are EBD-positive fibers. Scale bar: 100 μm.

Article Snippet: Primary antibodies. (i) Anti-mouse NCX3 exchanger; peptide DGNFTPLEGKEVDES from the mouse NCX3 protein sequence was used for rabbit immunization (Eurogentec, Herstal, Belgium).

Techniques: Staining, Injection

Journal:

Article Title: Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

doi: 10.1172/JCI200418688

Figure Lengend Snippet: Immunolocalization of NCX3 in sections of Ncx3+/+ and Ncx3–/– gastrocnemius muscles. (a and b) Both sarcolemma and sarcoplasm from Ncx3+/+ fibers are labeled with a polyclonal anti-NCX3 antibody (a); no immunoreactivity is detected in Ncx3–/– fibers (b). (c) Nerve ramifications inside the gastrocnemius muscle are also NCX3 positive. (d–f) Detection of NCX3 protein at the NMJ: triple labeling experiment with anti-NCX3 antibody (d), anti-VAChT antibody (e), and with the Alexa fluor 647-α-bungarotoxin (f) in a transverse section (15 mm) of Ncx3+/+ gastrocnemius muscle. Scale bars: 10 mm.

Article Snippet: Primary antibodies. (i) Anti-mouse NCX3 exchanger; peptide DGNFTPLEGKEVDES from the mouse NCX3 protein sequence was used for rabbit immunization (Eurogentec, Herstal, Belgium).

Techniques: Labeling

Journal:

Article Title: Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

doi: 10.1172/JCI200418688

Figure Lengend Snippet: Electromyographic findings in Ncx3–/– mice. (a) Recruitment curves of muscle responses evoked in the gastrocnemius muscle by stimulation of the sciatic nerve at increasing intensities. Mean CMAPs obtained in Ncx3+/+ (n = 5; filled circles) and Ncx3–/– (n = 5; open circles) mice are illustrated. (b and c) Effects of repetitive stimulation of the sciatic nerve at 10 (b) and 30 Hz (c) in Ncx3+/+ and Ncx3–/– mice. The graphs shown are representative of all the mice tested in the same group, except for Ncx3–/– mice at HRRS, where the graph is representative of one out of the 4/8 mice with an incremental CMAP. (d and e) Time course of the fifth response amplitude/first response amplitude ratios during LRRS (d) and HRRS (e) before and after exercise (20 minutes of repetitive stimuli) in Ncx3+/+ (n = 5; black bars) and Ncx3–/– (n = 5; gray bars) mice. (f) Ratios of the values illustrated in (d) and (e) (filled circles, Ncx3+/+ mice; open circles, Ncx3–/– mice). (g and h) MCDs expressed in microseconds and blocking rates expressed in percent in Ncx3+/+ mice (n = 22 fibers; MCDs, filled circles; blocking rate, open circles) and Ncx3–/– mice (n = 23 fibers; filled triangles, MCDs; open triangles, blocking rate). Statistics (in a, d–h): mean ± SEM; *P ≤ 0.05; **P ≤ 0.01.

Article Snippet: Primary antibodies. (i) Anti-mouse NCX3 exchanger; peptide DGNFTPLEGKEVDES from the mouse NCX3 protein sequence was used for rabbit immunization (Eurogentec, Herstal, Belgium).

Techniques: Blocking Assay

Journal:

Article Title: Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

doi: 10.1172/JCI200418688

Figure Lengend Snippet: Synaptic parameters recorded in hemidiaphragm neuromuscular preparations from Ncx3+/+ and Ncx3–/– mice. (a) Spontaneous quantal ACh release, measured as the frequency of MEPPs. The number of NMJs tested in each group is indicated in brackets. (b) Full-size EPP evoked at 0.1 Hz by a single nerve stimulus in a Ncx3–/– NMJ (left tracing) and after repetitive stimulation at 10 Hz (right tracing). The arrow indicates the increased MEPP frequency. Standard physiological solution supplemented with 2.2 μM μ-conotoxin GIIIB was used. The resting membrane potential during measurements was –74 ± 0.2 mV. (c) EPP amplitudes evoked at 50-Hz nerve stimulation (expressed as percent of the first EPP of each train, in sequential order). (d) Nerve-evoked, tetanus-twitch ratio at different stimulation frequencies in hemidiaphragm muscles from Ncx3+/+ and Ncx3–/– mice (n = 3). The resting membrane potential during recordings was –72 mV. Mean ± SD (a and d). *P ≤ 0.001.

Article Snippet: Primary antibodies. (i) Anti-mouse NCX3 exchanger; peptide DGNFTPLEGKEVDES from the mouse NCX3 protein sequence was used for rabbit immunization (Eurogentec, Herstal, Belgium).

Techniques:

Journal:

Article Title: Impaired neuromuscular transmission and skeletal muscle fiber necrosis in mice lacking Na/Ca exchanger 3

doi: 10.1172/JCI200418688

Figure Lengend Snippet: Behavior alterations in Ncx3–/– mice. (a) Average success score and (b) average falling score of Ncx3+/+ and Ncx3–/– mice in the wire test. (c) Time spent on the Rota-Rod. The number of animals used in each group is indicated in brackets. Mean ± SEM (c).

Article Snippet: Primary antibodies. (i) Anti-mouse NCX3 exchanger; peptide DGNFTPLEGKEVDES from the mouse NCX3 protein sequence was used for rabbit immunization (Eurogentec, Herstal, Belgium).

Techniques: