Immunoblot Analysis of Radiation-Induced Exosome Effects

For immunoblot, total proteins were extracted from cerebella (n = 2) at P3. Experimental groups included sham-injected (PBS) mice; mice injected with 0 Gy-plasma-derived exosomes (Evs SI (0 Gy); mice injected with 2 Gy PBI-plasma-derived exosomes (Evs PBI (2 Gy); mice injected with 2 Gy WBI-plasma-derived exosomes (Evs WBI (2 Gy); 2 Gy whole-body-irradiated mice (2 Gy WBI). All brain samples were collected at 6 h post-treatment. Proteins were extracted, normalized, separated, and immunoblotted as described [76 (link)]. To evaluate apoptosis, samples were incubated with Cleaved-caspase-3 (Asp 175) rabbit polyclonal antibody (dilution 1:1000; Cell Signaling Technology, Danvers, MA, USA). Monoclonal antibody against β-actin (dilution 1:10,000, Sigma-Aldrich, St. Louis, MO, USA) was used as a loading control. Specific proteins were visualized with ChemiDoc system XRS+ Biorad and quantified using ImageJ software.

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Pazzaglia S., Tanno B., De Stefano I., Giardullo P., Leonardi S., Merla C., Babini G., Tuncay Cagatay S., Mayah A., Kadhim M., Lyng F.M., von Toerne C., Khan Z.N., Subedi P., Tapio S., Saran A, & Mancuso M. (2022). Micro-RNA and Proteomic Profiles of Plasma-Derived Exosomes from Irradiated Mice Reveal Molecular Changes Preventing Apoptosis in Neonatal Cerebellum. International Journal of Molecular Sciences, 23(4), 2169.

Publication 2022

Cleaved-caspase-3 (Asp 175) rabbit polyclonal antibody Monoclonal antibody against β-actin ChemiDoc system XRS

Actin Antibody Apoptosis Body Brain Caspase 3 Cerebella Dilution Exosomes Mice Monoclonal antibody Plasma Proteins Rabbit

Corresponding Organization : National Agency for New Technologies, Energy and Sustainable Economic Development

Other organizations : Agostino Gemelli University Polyclinic, Istituti di Ricovero e Cura a Carattere Scientifico, Oxford Brookes University, Technological University Dublin, Helmholtz Zentrum München

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Variable analysis

independent variables

Sham-injected (PBS) mice
Mice injected with 0 Gy-plasma-derived exosomes (Evs SI (0 Gy))
Mice injected with 2 Gy PBI-plasma-derived exosomes (Evs PBI (2 Gy))
Mice injected with 2 Gy WBI-plasma-derived exosomes (Evs WBI (2 Gy))
2 Gy whole-body-irradiated mice (2 Gy WBI)

dependent variables

Cleaved-caspase-3 (Asp 175) protein expression

control variables

Total proteins extracted from cerebella (n = 2) at P3
Brain samples collected at 6 h post-treatment
Proteins extracted, normalized, separated, and immunoblotted as described [76]
Monoclonal antibody against β-actin (dilution 1:10,000, Sigma-Aldrich, St. Louis, MO, USA) used as a loading control

controls

Positive control: Not explicitly mentioned
Negative control: Sham-injected (PBS) mice

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