Journal: bioRxiv

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

doi: 10.1101/2024.06.26.600760

Figure Lengend Snippet: (A) Workflow of the global proteome survey in vitro . SH-SY5Y NT CTRL cells and shATM cells (knockdown, ATM-kd) were subjected to quantitative detection of peptides by LC-MS/MS. Protein abundance was observed for (i) unstressed conditions, (ii) upon osmotic stress by administration of CQ, and (iii) upon oxidative stress by administration of NaARS. Each comparison represents three NT CTRL vs. three shATM biological replicates. (B) Functional enrichment of biological processes, molecular functions and cellular components for downregulations (left, green and blue shades) and upregulations (right, orange shades) with a fold change of at least ± 2.0 determined by PANTHER classification system ( https://pantherdb.org/ ) when comparing NT CTRL and shATM conditions. (C) Volcano plot of selected downregulated proteins with fold change (calculated as log2 ratio) plotted against p-value (calculated as –log10). (D) Immunoblots for the quantification of secreted CHGA protein in cell culture medium with total protein staining as normalizer, and bar graphs with densitometric quantification of CHGA protein to total protein levels (n=3). Bar plots represent mean + SEM. Asterisks reflect significance: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, *** = p ≤ 0.0001; T = p ≤ 0.1, ns = non-significant. Abbreviations: CQ = chloroquine, NaARS = sodium arsenite, CHGA = chromogranin A.

Article Snippet: For co-immunoprecipitations, 800 - 1000 µg protein in 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 Signalling Technology, #5415) in binding buffer consisting of CEB fractionation buffer supplemented with HALT phosphatase inhibitors (Thermo Fisher Scientific) and cOmplete proteinase inhibitors (Roche) over night at 4 °C with head to tail rotation.

Techniques: In Vitro, Knockdown, Liquid Chromatography with Mass Spectroscopy, Comparison, Functional Assay, Western Blot, Cell Culture, Staining

Journal: bioRxiv

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

doi: 10.1101/2024.06.26.600760

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

Article Snippet: For co-immunoprecipitations, 800 - 1000 µg protein in 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 Signalling Technology, #5415) in binding buffer consisting of CEB fractionation buffer supplemented with HALT phosphatase inhibitors (Thermo Fisher Scientific) and cOmplete proteinase inhibitors (Roche) over night at 4 °C with head to tail rotation.

Techniques: Immunoprecipitation