Whole cell lysates/chromatin fractions were prepared and western blot analysis was performed as previously described12 (link). In brief, for chromatin extraction, cell pellets were lysed in buffer containing 10mM HEPES pH7.4, 10mM KCl, 0.05% NP-40 supplemented with a protease inhibitor cocktail (Complete EDTA-free, Roche Applied Science), 5 μM TSA, 5mM sodium butyrate, 1mM DTT, 1mM PMSF, and 0.2mM sodium orthovanadate. After incubation for 20min on ice, the lysates were centrifuged at 14,000 rpm, 10min at 4 °C. The supernatant was removed (cytosolic fraction) and the pellet (nuclei) was acid-extracted using 0.2N HCl by incubating 20min on ice. The lysate was further centrifuged at 14,000 rpm, 10min at 4 °C. The supernatant was neutralized in 1M Tris-HCl pH 8. Protein concentration was determined by Biorad protein assay. Western blots were performed using 8-15% gradient gels (Biorad). Primary antibodies were used as follows: anti-SIRT6 (Cell signaling #12486), anti-H3K9Ac (Millipore, 07-352), anti-H3K56Ac (Abcam, ab76307), anti-total H3 (Abcam, ab1791), anti-GLUT1 (Abcam, ab40084), anti-PDK1 (Cell signaling, #3820), anti-LDHA (Cell signaling, #2012S), anti-phospho-PDH (Abcam, ab92696), and anti-β-actin (Sigma, A5316). All uncropped and unprocessed scans are available in Source Data Figures 1 -4 .
Anti phospho pdh
Manufactured by Abcam
Anti-phospho-PDH is a primary antibody that specifically recognizes the phosphorylated form of the pyruvate dehydrogenase (PDH) enzyme. PDH is a key enzyme complex involved in the conversion of pyruvate to acetyl-CoA, a critical step in cellular energy metabolism. This antibody can be used to detect and quantify the phosphorylation status of PDH, which is an important regulatory mechanism for the enzyme's activity.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (2 mentions)
Complete edta free, by Roche (2 mentions)
Gradient gel, by Bio-Rad (2 mentions)
Anti h3k9ac, by Merck Group (2 mentions)
Anti total h3, by Abcam (2 mentions)
Anti h3k56ac, by Abcam (2 mentions)
Ab1791, by Abcam (2 mentions)
Ab40084, by Cell Signaling Technology (2 mentions)
Ab92696, by Merck Group (2 mentions)
Anti pdk1, by Cell Signaling Technology (2 mentions)
Anti sirt6, by Cell Signaling Technology (2 mentions)
Anti ldha, by Cell Signaling Technology (2 mentions)
Anti glut1, by Abcam (2 mentions)
2 protocols using anti phospho pdh
1
Chromatin Extraction and Western Blot Analysis
2
Chromatin Extraction and Western Blot Analysis
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Whole cell lysates/chromatin fractions were prepared and western blot analysis was performed as previously described12 (link). In brief, for chromatin extraction, cell pellets were lysed in buffer containing 10mM HEPES pH7.4, 10mM KCl, 0.05% NP-40 supplemented with a protease inhibitor cocktail (Complete EDTA-free, Roche Applied Science), 5 μM TSA, 5mM sodium butyrate, 1mM DTT, 1mM PMSF, and 0.2mM sodium orthovanadate. After incubation for 20min on ice, the lysates were centrifuged at 14,000 rpm, 10min at 4 °C. The supernatant was removed (cytosolic fraction) and the pellet (nuclei) was acid-extracted using 0.2N HCl by incubating 20min on ice. The lysate was further centrifuged at 14,000 rpm, 10min at 4 °C. The supernatant was neutralized in 1M Tris-HCl pH 8. Protein concentration was determined by Biorad protein assay. Western blots were performed using 8-15% gradient gels (Biorad). Primary antibodies were used as follows: anti-SIRT6 (Cell signaling #12486), anti-H3K9Ac (Millipore, 07-352), anti-H3K56Ac (Abcam, ab76307), anti-total H3 (Abcam, ab1791), anti-GLUT1 (Abcam, ab40084), anti-PDK1 (Cell signaling, #3820), anti-LDHA (Cell signaling, #2012S), anti-phospho-PDH (Abcam, ab92696), and anti-β-actin (Sigma, A5316). All uncropped and unprocessed scans are available in Source Data Figures 1 -4 .
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