Each reaction mixture contained 2 µg of recombinant GST-tagged TTP (wild type or mutants) served as substrates, 3 µl of 10X reaction buffer (New England Biolabs), 30 μM of ATP, and kinases including ERK2 (New England Biolabs), p38 alpha (SignalChem), RSK1 (SignalChem), and MK2 (SignalChem) in a nal volume of 30 µl. The kinases can be from cell extracts. 300 g of LPS-treated RAW264.7 whole cell extracts were incubated with GSH-Sepharose bound 2 g of GST-TTP in the buffer containing 20 mM HEPES, pH7.7, 75 mM NaCl, 0.5 mM MgCl 2 , 0.1 mM EDTA, 0.05 % Triton X-100, 0.5 mM DTT, 20 mMglycerolphosphate, 0.1 mM Na 3 VO 4 , 1 g/ml leupeptin, 1 g/ml papstatin A, 100 g/ml PMSF. The mixture was rotated at 4 o C for 3 hr and pelleted by centri gation at 10,000xg for 20 sec. After 4x1-ml washes in HEPES binding buffer (20 mM HEPES, pH7.7, 50mM NaCl, 2.5 mM MgCl 2 , 0.1 mM EDTA, 0.05 % Triton X-100), the beads were resuspended in 30 l for kinase assay. The reaction mixtures were incubated at 30°C for 30 minutes and stopped by adding one volume of protein sample buffer. Samples were subjected to SDS-PAGE for western blotting with anti-phospho-S316 and ponceau S staining.
P38 alpha
Manufactured by Sino Biological
P38 alpha is a recombinant protein that represents the catalytic domain of the p38 alpha mitogen-activated protein kinase (MAPK). P38 alpha is a member of the MAPK family and plays a crucial role in cellular responses to various environmental stresses and inflammatory cytokines.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using p38 alpha
1
Kinase Regulation of TTP Phosphorylation
2
Kinase-mediated Regulation of TTP Activity
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Each reaction mixture contained 2 μg of recombinant GST-tagged TTP (wild type or mutants) served as substrates, 3 μl of 10X reaction buffer (New England Biolabs), 30 μM of ATP, and kinases including ERK2 (New England Biolabs), p38 alpha (SignalChem), RSK1 (SignalChem), and MK2 (SignalChem) in a final volume of 30 μl. The kinases can be from cell extracts. 300 μg of LPS-treated RAW264.7 whole cell extracts were incubated with GSH-Sepharose bound 2 μg of GST-TTP in the buffer containing 20 mM HEPES, pH 7.7, 75 mM NaCl, 0.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100, 0.5 mM DTT, 20 mM β-glycerolphosphate, 0.1 mM Na3VO4, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 100 μg/ml PMSF. The mixture was rotated at 4 °C for 3 h and pelleted by centrifugation at 10,000×g for 20 s. After 4 × 1-ml washes in HEPES binding buffer (20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.05% Triton X-100), the beads were resuspended in 30 μl for kinase assay. The reaction mixtures were incubated at 30 °C for 30 min and stopped by adding one volume of protein sample buffer. Samples were subjected to SDS-PAGE for western blotting with anti-phospho-S316 and ponceau S staining.
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