Exploratory experiment designed to discover new patterns targeting PPP3CB in neuronal cell lines. Primary outcome: TFEB phosphorylation status and nuclear localization
This experiment investigated the molecular mechanism linking lysosomal membrane permeabilization to TFEB activation. The study examined calcium-dependent phosphatase PPP3/calcineurin activation following trehalose-induced lysosomal damage, leading to TFEB dephosphorylation and subsequent nuclear translocation. The researchers used calcium imaging, phosphatase activity assays, and immunofluorescence to demonstrate that lysosomal calcium release activates calcineurin, which then dephosphorylates TFEB, allowing its nuclear translocation and transcriptional activity.
Calcium imaging, immunofluorescence microscopy, western blotting for phospho-TFEB, subcellular fractionation, phosphatase activity assays
Lysosomal calcium release would activate calcineurin, leading to TFEB dephosphorylation and nuclear translocation
Demonstration of calcium-dependent TFEB dephosphorylation and nuclear translocation that can be blocked by calcineurin inhibitors
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