Rabbit anti hif 1α

Mouse anti-c-myc monoclonal antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Mouse anti-β-actin was purchased from Millipore Corporation (Billerica, MA, USA). Rabbit anti-HIF-1α, rabbit anti-glut1, rabbit anti-LDHA, rabbit anti-pyruvate kinase isozyme M2 (PKM2), rabbit anti-2-oxoglutarate dehyrogenase E1 component (OGDH), and rabbit anti-glutaminase (Gls) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against ARV σA and p17 proteins were from our laboratory stocks [8 (link),16 (link)]. The secondary antibodies, including goat anti-mouse IgG (H + L) and goat anti-rabbit IgG (H + L) HRP conjugate and rhodamine-labeled affinity purified antibody to mouse IgG (H + L) were purchased from Kirkegaard and Perry Laboratories (Washington, DC, USA).

Cultured cells were harvested after the reported treatments (as described as above), washed with cold PBS on ice, and lysed with RIPA lysis buffer for protein extraction. After being denatured at 100°C for 10 min, aliquots of protein samples (20 μg) were separated by electrophoresis on an SDS-polyacrylamide gel (4% pycnotic gel and 10% separation gel, Bio-Rad Laboratories, Inc), then transferred onto a polyvinylidene difluoride membrane (Millipore Corporation), blocked for 1 h with 5% skimmed milk in 0.05% Tris-buffered saline/Tween (TBST), and incubated with a specific primary antibody (diluted 1:1000 in blocking buffer for the rabbit anti-HIF-1α; Cell Signaling Technology, London, United Kingdom), 1:500 for the rabbit anti-CA9 (Abcam, Cambridge, United Kingdom), 1:1000 for the mouse anti-VEGF, 1:1000 for mouse anti-Glut 1, and 1:1000 for mouse anti-β-actin (Abcam) overnight at 4°C. Then, the membrane was washed 3× with TBST at room temperature for 10 min and incubated with a secondary antibody (HRP-conjugated anti-mouse IgG or anti-rabbit IgG; 1:5000, Abcam) for 1 h. The membrane was detected by the enhanced chemiluminescence system (ECL Advance, Amersham Biosciences) after several washes, and the protein image was recorded using a BIO-RAD ChemiDoc XRS and analyzed using Quantity One software (Bio-Rad, Hercules, CA, United States).

The primary antibodies used for western blot are as follows: rabbit anti-HIF-1α (1:1000, Cell Signaling Technology, RRID: AB_2799095), and rabbit anti-LDHA (1:5000, Abcam, RRID: AB_2889291). For western blotting, horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000, Sigma-Aldrich) were used. The primary antibodies used for immunofluorescent staining are as follows: rabbit anti-CD8α (1:200, Abcam, RRID: AB_2890649), rabbit anti-CD4 (1:100, Abcam, RRID: AB_2686917), rabbit anti-GFAP (1:200, Cell Signaling Technology, RRID: AB_2799963), and rabbit anti-Iba1 (1:200, Cell Signaling Technology, RRID: AB_2820254). The secondary antibodies used for immunohistochemical staining were bought from Invitrogen.