Sc 13515

Immunnohistochemical staining for BCL9 was performed by using the standard histological procedure described in the manual for Histostain-Plus IHC Kit, DAB (Phygene). BCL9 staining was scored according to two investigators with previous protocols as negative (−), weak positive (+), and strong (++). The results were analyzed by standard light microscopy. Scoring was assessed blindly with respect to the histologic grade of HCC specimens. Immunofluorescence staining was performed with primary and secondary antibodies diluted in 10% BSA, and the nucleus was stained by DAPI (Sigma). The antibody against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), antibody against HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), antibody against BCL9 (ab37305, Abcam, 1:500 dilution) and antibody against β-actin (A5441, Sigma) were used in this study. The antibodies are anti-rabbit in this study. All fluorescent secondary antibodies were used at a dilution of 1:200 for 30 min (invitrogen). Quantification of immunofluorescence was performed using NIH Image J.

All biopsy specimens were obtained from that part of the skin flap, and each biopsy specimen was harvested with a small pair of microsurgical scissors with a sample size of 1cm³. Protein was extracted from specimens with RIPA buffer containing protease inhibitors, separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. The membranes were incubated overnight at 4°C with anti-Hif-1a Ab (1:1000 dilution; sc-13515, Santa Cruz Biotechnology, USA) and anti-β-actin Ab (1:1,000 dilution; CP01; Calbiochem, San Diego, CA, USA), followed by incubation with corresponding HRP-conjugated secondary antibodies. Signals were detected in a sensitive digital imaging equipment (Image Quant LAS 4000 mini; GE Healthcare Bio-Sciences AB, Uppsala, Sweden) using a commercial ECL detection kit (Millipore, Billerica, MA).

Standard Western-blot assays were used to analyze the levels of protein. Antibodies against HIF-1α (sc-13515, Santa Cruz Biotechnology, 1:500), anti-HIF-2α (sc-13596, Santa Cruz Biotechnology, 1:500), anti-BCL-9 (ab37305, Abcam, 1:500 dilution) and anti–β-actin (A5441, Sigma) antibodies were used in this study.

The cell or tissue samples were lysed using RIPA Buffer (P0013B, Thermo Fisher Scientific, USA) containing protease and phosphatase inhibitors (78425, Thermo Fisher Scientific, USA; B15001, B15002, Bimake, USA) and protein extraction was performed according to the manufacturer's instructions. Concentration of the protein was determined using the BCA protein assay kit (23225, Thermo Fisher Scientific, USA). Denatured protein diluted to an equal concentration was separated by SDS-PAGE. Subsequently, the protein was transferred to a PVDF membrane at a constant current of 300 mA. After blocked in 5% skim milk for 1 h, the membrane was incubated overnight with the primary antibody: rabbit anti-GFAP antibody (1:1000; 16825-1-AP, Proteintech, USA), mouse anti-HIF-1α antibody (1:200; SC-13515, Santa Cruz, USA), rabbit anti-IBA1 antibody (1:1000; ab178846, Abcam, USA), mouse anti-TH antibody (1:1000; 22941, Immunostar, USA), mouse anti-β-actin antibody (1:1000; SC-47778, Santa Cruz, USA). The next day, after washing, the membrane was incubated with the corresponding secondary antibody for 1 h, and the band of interest was detected using the Odyssey infrared imaging system (Li-Cor, USA). The relative level of protein was quantified using ImageJ software (NIH, Bethesda, MD).

The protein levels of CPT1A, Flag-pep-AKR1C2, pep-AKR1C2, YAP, p127-YAP, VEGFC and HIF-1α were assessed by western blotting analysis and samples were normalized to GAPDH. For animals, mice were firstly sacrificed with carbon dioxide asphyxiation. Protein extraction was blocked with PBS-5% fat-free dried milk at room temperature for 1 h and incubated at 4 °C overnight with anti-CPT1A (1:1000; Abcam, Cambridge, UK; ab234111), anti-Flag (1:5,000; Abcam, Cambridge, UK; ab205606), anti-YAP (1:1000, Abcam; ab205270), anti p127-YAP (1:1000, Abcam; ab76252), anti-pep-AKR1C2 (1:1000, CST, 13035 S), anti-VEGFC (1:1000, Abcam; ab9546), anti-HIF-1α (Santa Cruz, sc-13515) anti-TSG101 (1:200; Santa Cruz, sc-7964), anti-Alix (1:1000, Abcam, ab275377), anti-CD9 (1:1000, Abcam, ab223052), anti-H3 (1:1000, Abcam, ab1791) and anti-GAPDH (1:3000, Santa Cruz, sc-365062) antibodies respectively.

Retina and myelinated ONs were collected in T-PER buffer with HALT protease and phosphatase inhibitors, then disrupted with a Branson Sonifier to create a protein lysate. After centrifugation for 15 min at 15,000 rpm, the supernatant was collected. Total protein concentration was measured by a Bicinchoninic Acid (BCA) assay kit. Proteins were analyzed by capillary tube-based electrophoresis immunoassay using the Wes and normalized to total protein in the sample. Wes is a Protein Simple instrument that separates proteins by an electrical charge in capillary tubes and allows binding of primary antibody then protein visualization within the capillary. Protein analysis for GLUT1 (1:50, rabbit, Novus Biologicals, Littleton, CO, USA, NB110-39113), GLUT3 (1:25, mouse, R & D systems, Minneapolis, MN, USA, NAB1415), HIF-1α (1:1000, mouse, Santa Cruz, Dallas, TX, USA, sc-13515), and HIF-2α (mouse, 1:1000, Santa Cruz, Dallas, TX, USA, sc-13596) was repeated at least three times with biological replicates [7 (link),19 (link)].

Immunohistochemical staining was performed according to previous description [19 (link)]. Monoclonal mouse anti-human CD34 antibody (1:100 dilution, sc-65261; Santa Cruz) and monoclonal mouse anti-human HIF-1α (1:100 dilution; sc-13515, Santa Cruz) were incubated with the tumor sections overnight. After being washed with PBS for 3 times, the tumor sections were incubated with rabbit anti-mouse biotinylated secondary antibody at room temperature for 15 min, followed by incubating with horseradish peroxidase (HRP)-conjuncted streptavidin for another 30 min at room temperature. Diamino-benzidine (DAB) assay was used to detect the immunoactivity.