Cuspin-1

Kinase activity assays measure the ability of kinases to transfer phosphate groups from ATP to specific substrates, while phosphorylation assays detect the presence and levels of phosphorylated proteins. Common methods include radiolabeled ATP incorporation (e. g. ,), ADP release detection via bioluminescence (e. g. ,[3]), enzyme-linked immunosorbent assays (ELISA) for phospho-specific epitopes (e. g. ,[6]), and microtiter-based formats for high-throughput screening (e. g. ,[8]). The ADP-Glo assay quantifies kinase activity by measuring ADP produced during phosphorylation using a luciferase-based system. Radiometric assays involve autoradiography or scintillation counting after incorporation of ³²P-labeled ATP into substrate proteins. ELISA-based approaches rely on phospho-specific antibodies to detect activated kinases in cell lysates or purified samples.

By lysing cells, releasing RNA, and removing impurities such as proteins and DNA, high-purity RNA products are finally obtained. The commonly used traditional method is the guanidine isothiocyanate/phenol/chloroform method (Trizol), which is suitable for a variety of animal materials including animal tissues, microorganisms, cultured cells, etc., and most plant materials.

Western blotting (WB) is a commonly used experimental method in molecular biology, biochemistry, and immunogenetics for identifying and quantifying target proteins. It combines gel electrophoresis with immunoassay, enabling researchers to analyze protein expression, post-translational modifications, and molecular weight.

Kinase activity assays measure transfer of phosphate from ATP to a protein or peptide substrate, generating phosphorylated substrate, ADP, or incorporated radiolabeled phosphate as the readout; phosphorylation assays measure site-specific phosphorylation in cells or tissues as a proxy for kinase-pathway activation, inhibition, or substrate regulation. Phosphorylation can be detected by phospho-specific Western blot, immunoprecipitation kinase assay, phospho-immunofluorescence, phospho-flow cytometry, luminescent ADP detection, radiolabeled ATP incorporation, or reporter-based pathway assays, and these readouts can be applied to cancer cells, primary neurons, mouse tumors, organoids, inflammatory macrophages, ferroptosis studies, and mitophagy studies when the kinase target is biologically relevant.

PINK1/Parkin-mediated mitophagy pathway is a mitochondrial quality-control signaling axis in which mitochondrial depolarization stabilizes PINK1 on damaged mitochondria, activates Parkin recruitment and E3 ubiquitin ligase activity, promotes ubiquitination of outer mitochondrial membrane proteins, recruits selective autophagy adaptors, and drives lysosomal degradation of damaged mitochondria. In neurological disease research, this pathway is experimentally important because neurons, especially dopaminergic neurons, are highly dependent on mitochondrial integrity, and defective mitochondrial turnover can lead to mitochondrial dysfunction, oxidative stress, impaired neuronal survival, α-synuclein accumulation, and neuroinflammatory damage-associated signals. The genetic disease link is strongest in Parkinson’s disease because mutations in PRKN/parkin cause autosomal recessive juvenile parkinsonism, mutations in PINK1 cause hereditary early-onset Parkinson’s disease, and Drosophila studie