Enolase 2

Cells were lysed in modified RIPA buffer (50 mM Tris–HCl (pH 7.5), 150 NaCl, 10 mM β‐glycerophosphate, 1% NP‐40, 0.25% sodium deoxycholate, 10 mM sodium pyrophosphate, 30 mM sodium fluoride, 1 mM EDTA, 1 mM vanadate, 20 μg/ml aprotinin, 20 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Whole‐cell lysates were resolved by SDS–PAGE on 4–15% gradient gels and blotted onto nitrocellulose membranes (Bio‐Rad). Membranes were blocked overnight and then incubated sequentially with primary and either HRP‐conjugated (Pierce) or IRDye‐conjugated secondary antibodies (Li‐Cor). Blots were imaged using the Odyssey Infrared Imaging System (Li‐Cor). Protein levels were quantitated using ImageJ (http://imagej.nih.gov/ij/). Primary antibodies used for Western blot analysis included hexokinase 1 (2024, Cell Signaling Technology), hexokinase 2 (2867, Cell Signaling Technology), p53 (NB200‐103, Novus Biologicals), and enolase 2 (8171, Cell Signaling Technology).

Compounds utilized in our studies, including the small molecule inhibitors panobinostat, marizomib, 2‐DG, IACS‐010759, lonidamine, antimycin A, FCCP, and Gboxin, were purchased from Selleck Chemicals (Houston, TX, USA). Fluorescent probes were purchased from Invitrogen/Molecular Probes (Eugene, OR, USA) unless otherwise stated. The following antibodies were used: BiP (#3177), cleaved caspase 3 (#9664), cleaved caspase 7 (#8438), caspase 8 (#9746), Enolase‐2 (#24330), phospho‐H2AX (#80312), GAPDH (#2118), Hexokinase‐2 (#2867), PGC‐1α (#2178), RAD51 (#8875), and β‐Actin (#4970); all were from Cell Signaling Technology Inc., (Beverly, MA, USA).