Dephosphorylation of Activated TFEB

Phosphorylated FLAG-TFEB was obtained by immunoprecipitation from fed HeLa cells. Protein-G beads containing FLAG-TFEB were incubated for 40 min at 30°C in 24 µl of corresponding phosphatase reaction buffer (20 mM HEPES, pH 7.5, 10 mM MgCl₂, 2 mM DTT, protease inhibitors with 1 mM CaCl₂ or 10 mM EGTA) containing purified Calcineurin (100U; ENZO Life Sciences) and Calmodulin (4 mM; ENZO Life Sciences). Samples were eluted by adding 8 µl of 4X Laemmli buffer, resolved by SDS–PAGE and analysed by immunoblotting. Antibodies anti-p-Ser 14-3-3 binding motif^{10 (link)}, anti-P-Ser142-TFEB^{9 (link)} and TFEB (Cell Signaling Technologies) were used to detect phosphorylated and total TFEB forms, respectively.

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Medina D.L., Di Paola S., Peluso I., Armani A., De Stefani D., Venditti R., Montefusco S., Scotto-Rosato A., Prezioso C., Forrester A., Settembre C., Wang W., Gao Q., Xu H., Sandri M., Rizzuto R., De Matteis M.A, & Ballabio A. (2015). Lysosomal calcium signaling regulates autophagy via calcineurin and TFEB. Nature cell biology, 17(3), 288-299.

Publication 2015

Calcineurin Calmodulin anti-p-Ser 14-3-3 binding motif10 anti-P-Ser142-TFEB9

14 3 3 Antibodies Buffer Calcineurin Calmodulin Egta Hela cells Hepes Immunoprecipitation Laemmli buffer Mgcl2 Phosphatase Protease inhibitors 1 Protein g Sds page Tfeb

Corresponding Organization : Telethon Institute Of Genetics And Medicine

Other organizations : University of Padua, University of Michigan–Ann Arbor, Federico II University Hospital

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Variable analysis

independent variables

Calcineurin (100U; ENZO Life Sciences)
Calmodulin (4 mM; ENZO Life Sciences)

dependent variables

Phosphorylated FLAG-TFEB
Phosphorylation of TFEB at Ser142
Phosphorylation of TFEB at the 14-3-3 binding motif

control variables

Incubation time: 40 min
Incubation temperature: 30°C
Phosphatase reaction buffer: 20 mM HEPES, pH 7.5, 10 mM MgCl2, 2 mM DTT, protease inhibitors
Presence of 1 mM CaCl2 or 10 mM EGTA in the phosphatase reaction buffer

positive controls

None specified

negative controls

None specified

Annotations

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