Sc 2005

Total cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer (Sigma-Aldrich) supplemented with a protease inhibitor cocktail (Sigma-Aldrich) as well as Phosphatase Inhibitor Cocktails 2 and 3 (Sigma-Aldrich). Samples were separated on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were hybridized with rabbit anti-adipose triglyceride lipase (ATGL; 2138, 1:1,000; Cell Signaling Technology), rabbit anti-phospho-hormone-sensitive lipase (HSL; Ser563; 4139, 1:1,000; Cell Signaling Technology), rabbit anti-HSL (4107, 1:1,000; Cell Signaling Technology), mouse anti-LXRα (ab41902, 1:1,000; Abcam), rabbit anti-LXRβ (ab28479, 1:500; Abcam), and mouse anti-GAPDH (MAB374, 1:80,000; Millipore) antibodies at 4°C overnight to detect expression, followed by 1-h incubation at room temperature with goat anti-mouse (sc-2005, 1:4,000; Santa Cruz Biotechnology) and goat anti-rabbit (P0448, 1:2,000; Dako) peroxidase-conjugated antibodies. Proteins were detected by chemiluminescence (Millipore).

Cell or tissue lysates with equal amounts of protein (40 μg) were mixed with loading dye, boiled for 5 min, separated on a denaturing 12.5-% SDS-polyacrylamide gel and transferred to Amersham™ Hybond™ 0.45 μm PVDF membrane (GE Healthcare, UK). The membrane was blocked in 5% dry milk or BSA in TBS buffer with 0.1% Tween (TBS-T) for 1 h and incubated overnight with antibodies against glutathione-S-transferase A3 (ab175246, Abcam), glutathione-S-transferase P1 (PA5-29601, Invitrogen), glutathione reductase (ab16801, Abcam), superoxide dismutase 2 (PA5-30604, Invitrogen), NAD(P)H quinone oxidoreductase 1 (PA5-19624, Invitrogen), pro-caspase 3 and active caspase 3 (ab13847, Abcam), or β-actin (4967, Cell Signaling) as a reference. Then, the membrane was washed twice with TBS-T and incubated with HRP-conjugated secondary antibody (ab6721, Abcam or sc-2005, Santa Cruz Biotechnology) at room temperature for 1 h, followed by several washings with TBS-T and deionized water. Protein bands were visualized by ChemiDoc XRS+ imaging system (Bio-Rad Laboratories, Hercules, CA) using chemiluminescence mode.

Western blotting was performed as previously described^{22 (link), 23 (link), 46 (link)}. The lysates from the xenografts or cell lines with different treatments were homogenized in RIPA lysis buffer using a homogenizer. The supernatants were collected by centrifugation (12,000 rpm at 4 °C for 25 min; Beckman GS-6R). Protein was resolved by electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were probed with monoclonal HIF-1α (NB100–105, Novus biological, Littleton, CO, USA), Bax (2772 S, Cell signaling, Boston, MA, USA), Bcl-2 (3498S, Cell signaling), caspase-3 (9661S, Cell signaling), PARP (5625, Cell signaling), and PCNA (18197, Abcam, Cambridge, MA, USA), as well as p21 (Sc817, Santa Cruz, CA, USA), CDK4 (Sc70831, Santa Cruz), SP1 (ab27595, Abcam), HK2 (2772S, Cell signaling), PKM2 (4053S, Cell signaling), LDH-A (3582S, Cell signaling), and PDK1 (3820 S, Cell signaling). Following incubation with the primary antibody overnight at 4 °C, membranes were washed with Tris-buffered saline (pH 7.2) containing 0.05% Tween-20 and subsequently incubated with horse radish peroxidase (HRP)-conjugated anti-mouse (Sc-2005, Santa Cruz) or anti-rabbit (Sc-2357, Santa Cruz) secondary antibody for 1 h at room temperature. Bands of interest were analyzed using the ChemiDocTM Touch Imaging System (BIO-RAD, Hercules, CA, USA).