Anti pdk1

Whole cell lysates/chromatin fractions were prepared and western blot analysis was performed as previously described^{12 (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.

Inactive Akt1, active Akt, active PDK1 and ATP/Mg²⁺ cocktail were purchased from Upstate Cell Signaling Solutions. Mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) was purchased from Calbiochem. 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (18:0,22:6-PS), 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (18:0, 22:6-PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (16:0, 18:1-PC), and 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol-3,4,5-trisphosphate (18:0, 20:4-PIP₃) were purchased from Avanti Polar Lipids. Sequencing grade modified trypsin was purchased from Promega. Anti-phospho-Akt (T308) (Cat#: 2965), anti-phospho-Akt (S473) (Cat#: 9271), anti-Akt (Cat#: 9272), anti-protein kinase C zeta (PKCζ)(Cat#: 9368), anti-phospho-PKCζ(T410) (Cat#: 9378), anti-phospho-PDK1(S241) (Cat#: 3061), anti-PDK1 (Cat#: 3062), and anti-GAPDH (Cat#: 2118) antibodies were purchased from Cell Signaling Technology. Anti-phospho-Akt (T34) (Cat#: 23509) was obtained from Abcam. A dilution of 1:1000 was used in western blotting for all of these antibodies. Insulin-like growth factor-1 (IGF-1) was purchased from PeproTech. PBS without Ca²⁺ was purchased from Quality Biological, Inc. Other reagents were purchased from Sigma or Quality Biological, Inc.

Mouse skeletal muscle samples were fixed in 4% paraformaldehyde overnight at 4°C, followed by dehydrated in PBS containing 30% sucrose. Mouse skeletal muscle samples were embedded in paraffin, 10mm-thick paraffin sections were used for immunofluorescence staining. The primary antibodies used were: rabbit anti-AKT (1:100, Cell Signaling Technology, 4685), anti-PDK1 (1:100, Cell Signaling Technology, 5662), anti-GLUT4 (1:100, Abcam, ab654), mouse anti-E-cadherin (1:100, Cell Signaling Technology, 14472). The secondary antibodies (1:1000, Thermo Fisher Scientific) used were: Alexa Fluor 488 donkey anti-mouse and Alexa Fluor 555 donkey anti-rabbit. Sections were examined suing a Zeiss LSM710 confocal microscope as mentioned above.