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 motif10 (link), anti-P-Ser142-TFEB9 (link) and TFEB (Cell Signaling Technologies) were used to detect phosphorylated and total TFEB forms, respectively.
Anti p ser142 tfeb9
Manufactured by Cell Signaling Technology
The Anti-P-Ser142-TFEB antibody is a research tool used to detect the phosphorylation of TFEB (Transcription Factor EB) at serine 142. TFEB is a transcription factor involved in the regulation of lysosomal biogenesis and autophagy. The phosphorylation of TFEB at serine 142 is an important post-translational modification that can influence its subcellular localization and activity.
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2 protocols using anti p ser142 tfeb9
1
Dephosphorylation of Activated TFEB
2
Dephosphorylation of Activated TFEB
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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 motif10 (link), anti-P-Ser142-TFEB9 (link) and TFEB (Cell Signaling Technologies) were used to detect phosphorylated and total TFEB forms, respectively.
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