atm antibody (5c2)  (Bio-Techne corporation)

Journal: Neurobiology of disease

Article Title: The ataxia-telangiectasia disease protein ATM controls vesicular protein secretion via CHGA and microtubule dynamics via CRMP5.

doi: 10.1016/j.nbd.2024.106756

Figure Lengend Snippet: Fig. 5. ATM interacts with CRMP5 and Spastin (SPAST) in vitro. (A) Immunoprecipitation of ATM by anti-ATM (5C2) antibody in unstressed and Taxol-treated cells, with detection of CRMP5 and SPAST as interacting proteins. (B) Immunoprecipitation of CRMP5 protein in unstressed and Taxol-treated cells, with detection of ATM and SPAST as interacting proteins. Abbreviations: SPAST = Spastin.

Article Snippet: For co-immunoprecipitations, 800–1000 μg protein from cytoplasmic subcellular fractions of SH-SY5Y cells were incubated with either 10 μg anti-ATM antibody (Novus Biologicals, Littleton, Colorado, USA, NB100–220) or 10 μg of mouse IgG1 isotype control antibody (Cell Signaling Technology, #5415) in binding buffer consisting of CEB fractionation buffer supplemented with HALT phosphatase inhibitors (Thermo Fisher Scientific) and cOmplete protease inhibitors (Roche) overnight at 4 ◦C with head to tail rotation.

Techniques: In Vitro, Immunoprecipitation

Journal: Neurobiology of disease

Article Title: The ataxia-telangiectasia disease protein ATM controls vesicular protein secretion via CHGA and microtubule dynamics via CRMP5.

doi: 10.1016/j.nbd.2024.106756

Figure Lengend Snippet: Fig. 8. Proposed pathway of novel cytoplasmic ATM-mediated signaling events. CHGA protein levels and secretion is reduced compared to normal cells with neural origin (right panel) and CRMP5 protein levels as well as phosphorylation at Ser538 are diminished upon ATM loss (left panel). Given the crucial role of CHGA in dense-core granule biogenesis (Koshimizu et al., 2010), we propose that reduced CHGA in granules in the trans-Golgi network (TGN) diminishes budding of large dense core vesicles (LDCV) from TGN membrane resulting in reduced transport of LDCVs along the axon and reduced secretion of LDCVs to the extracellular space. This likely results in reduced neuropeptide and neurotrophic signaling. Concomitant CRMP5 protein loss causes increased polymerization of microtubules, probably via reduced binding and sequestration of tubulin heterodimers, shifting the microtubule equilibrium from dynamic towards stabilized, thereby causing neurite retraction. Abbreviations: CHGA = Chromogranin A; CRMP5 = Collapsin Response Mediator Protein 5; ER = endoplasmic reticulum; INA = Internexin-alpha; LDCV = large dense-core vesicle; NFL = Neurofilament light chain; TANC2 = Tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 protein.

Article Snippet: For co-immunoprecipitations, 800–1000 μg protein from cytoplasmic subcellular fractions of SH-SY5Y cells were incubated with either 10 μg anti-ATM antibody (Novus Biologicals, Littleton, Colorado, USA, NB100–220) or 10 μg of mouse IgG1 isotype control antibody (Cell Signaling Technology, #5415) in binding buffer consisting of CEB fractionation buffer supplemented with HALT phosphatase inhibitors (Thermo Fisher Scientific) and cOmplete protease inhibitors (Roche) overnight at 4 ◦C with head to tail rotation.

Techniques: Phospho-proteomics, Membrane, Binding Assay