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2 protocols using ab15311

1

Phosphorylated PDH-E1α and Glucose Transporters

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Cells were lysed in a low detergent buffer (1% Triton, 0.1% SDS) for one hour with protease and phosphatase inhibitors (Sigma-Aldrich). Nitrocellulose membranes were hybridized with anti-phospho S232-PDH-E1α (Millipore AP1063), total PDH-E1α antibodies (Abcam ab110334), Glut1 rabbit monoclonal (abcam, ab115730), Glut3 rabbit polyclonal (abcam, ab15311), actin (Cell Signaling, 4970S), cMyc (Cell Signaling, 179) or Hif1α (Cayman Chemicals 10006421).
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2

Investigating Cellular Signaling Pathways

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Cell lysates from H460, A549, H1975, and HCC827 cells were prepared 24 h after treatment with DMSO, Milciclib (1 or 10 μM), THZ1 (10 μM), or LDC4297 (10 μM); 16 h after treatment with 1× PBS or Deferoxamine (100 μM); or 72 h after transfection with shRNA or an overexpression plasmid. Uncropped immunoblots are presented in Supplementary Fig. 29.
The following antibodies were used: HIF-1α (Novus Biologicals; NB100-105; 1:1000 dilution), GLUT1 (Millipore Sigma; 07-1401; 1:500 dilution), GLUT3 (Abcam; ab15311; 1:1000 dilution), Hexokinase 1 (Cell Signaling; C35C4; 1:1000 dilution), Hexokinase 2 (Cell Signaling; C64G5; 1:1000 dilution), CDK2 (Cell Signaling; 78B2; 1:1000 dilution), CDK4 (Cell Signaling; D9G3E; 1:1000 dilution), CDK7 (Cell Signaling; MO1; 1:1000 dilution), TRKA (Cell Signaling; 12G8; 1:1000 dilution), PIK3CA (Cell Signaling; C73F8; 1:1000 dilution), PTEN (Cell Signaling; 9559; 1:1000 dilution), Phospho-T170 CDK7 (Abcam; ab155976; 1:1000 dilution), Rpb1 NTD (Cell Signaling; D8L4Y; 1:1000 dilution), Phospho-S5 Rpb1 (Cell Signaling; D9N5I; 1:1000 dilution), PKCι (BD Biosciences; Clone 23; 1:1000 dilution), TXNIP (Cell Signaling; D5F3E; 1:1000 dilution), and Beta Actin (Imgenex; IMG-5142; 1:1000 dilution). Immunoblots were analyzed using an Odyssey Imaging System (LI-COR).
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