Ab131263

Less than 100 mg of snap‐frozen tissue was lysed in RIPA buffer supplemented with protease inhibitor cocktail (Merck‐Sigma, Gillingham, UK). Tissue was homogenised using a Polytron tissue homogeniser for 30 s followed by centrifugation, for 15 min at 4 °C, to pellet tissue debris. Lysates were subjected to SDS‐PAGE and transferred to nitrocellulose membranes (Amersham Biosciences, Amersham, UK) for western blotting. Blots were probed for β3 integrin (antibody a kind gift from Barry Coller, Rockefeller University; 1:500), succinate dehydrogenase (ab178423, 1:1000; Abcam, Cambridge, UK), pyruvate dehydrogenase (ab131263, 1:1000; Abcam), aconitase (ab129069, 1:10 000; Abcam), and GAPDH (AB2302; Merck, Hoddeston, UK). Densitometric readings of band intensities were obtained using ImageJ software (NIH, USA).

Tissues were homogenized with liquid nitrogen. Cells and tissues were then lysed by lysis buffer for 10 min on ice and then, centrifuged at 12,000 rpm for 15 min at 4°C. Supernatants were mixed with loading buffer and underwent SDS-PAGE to separate proteins. Proteins were then transferred into the PVDF membrane. The membranes were blocked by 5% nonfat milk in Tris-buffered saline (TBS) and then, incubated with anti-β-Actin (1 : 1000, ab6276, Abcam, USA), anti-HIF1α (1 : 500, ab179483, Abcam, USA), anti-IDH2 antibodies (1 : 500, ab131263, Abcam, USA), and anti-Hydroxy-HIF1α (1 : 500, 3434, Cell Signaling Technology, USA) at 4°C overnight. After being washed for 3 times by 0.5% TBST, membranes were incubated with second antibodies at a dilution of 1 : 4000 at room temperature for 2 hours then washed by 0.5% TBST for 3 times. Blots were then quantified by electrochemiluminescence and visualized by Gel Imaging System (GelDoc-It310, UVP, USA).

Approximately 40 μg protein per sample was separated on 12% SDS (sodium dodecyl sulfate) polyacrylamide gels. Proteins were transferred onto 0.45 μm PVDF membranes (IPVH00010; Millipore, Boston, Massachusetts, USA) and blocked with blocking buffer (Beyotime, Jiangsu, China). The membranes were incubated with primary antibodies to SCP2 (non-specific lipid-transfer protein 2, ab140126; Abcam, Cambridge, MA, USA), IDH2 (isocitrate dehydrogenase 2; ab131263, Abcam), SLC7A8 [(solute carrier family 7 (amino acid transporter, L system), member 8, ab75610, Abcam)], COL4A2 (collagen, type IV, alpha 2, sc-70,243, Santa Cruz biotechnology; Cambridge, MA, USA), and β-actin (Beyotime). After washing with TBST [tris-buffered saline containing 0.02% (v/v) Tween-20] three times, the membranes were incubated with goat anti-rabbit IgG or goat anti-mouse IgG secondary antibodies conjugated with horseradish peroxidase (Beyotime), incubated with ECL (electrochemiluminescence) Western Blotting Substrate Kits (Beyotime), and finally visualized with a Kodak Image Station 2000MM (Kodak Molecular Imaging Systems, New Haven, USA). The relative intensities of bands were calculated with ImagePro Plus 6.0 software (Media Cybernetics, Washington, MD, USA) using β-actin as the reference protein.

Protein levels of interest were determined by Western blot analysis according to a protocol described elsewhere [20 (link)]. Cells were treated with NTG in the 6-well plate, and the whole-cell lysates were quantified with a BCA kit (P0012, Beyotime, Shanghai, China). The protein sample (30 μg/sample) was loaded in each lane of the gel. The primary antibodies to pAMPK (α1T183 + α2T172, ab133448), pPDE1a (Ser297, ab177461), Citrate synthase (ab129095), IDH2 (ab131263), MDH2 (ab181873), Complex I (NDUFB8, ab110242), Complex II (ab110410), Complex III (UQCRC2, ab14745), Complex IV (COX IV, ab16056) and β-tubulin (ab6046) were obtained from Abcam (Cambridge, MA, USA). Antibody to ATP5A1 (495240) was from Invitrogen (Camarillo, CA, USA). β-tubulin was used as the internal control. The signal was collected with a Chemiluminescence gel imager (Amersham Imager 600, GE Healthcare, Chicago, IL, USA) and the images were quantified with the Image J software.