Ab82832

Denatured protein lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrophoretic transfer to polyvinylidene difluoride (PVDF) membranes using a Mini Trans-Blot Electrophoretic Transfer Cell (BioRad, Hercules, CA, USA). The PVDF membranes were then blocked with 5% (w/v) skim milk in 1× TBS-T solution (Tris-buffered solution (TBS) + 0.1% Tween 20) and incubated with primary antibodies against BCL2A1 (ab45413) and HIF-1α (ab82832) (Abcam, Cambridge, MA, USA), hydroxyl-HIF-1α (#3434), NF-κB p65 (#8242), IκB α (#4814), p-IκB α (#2859), Cytochrome c (#11940), cleaved-PARP (#5625) and cleaved-Caspase 3 (#9664) (Cell Signaling Technology, Danvers, MA, USA), and β-actin (Sigma-Aldrich, St. Louis, MI, USA) in 1% BSA in TBS-T overnight at 4 °C. The membranes were then incubated with mouse or rabbit polyclonal secondary antibodies (Amersham Pharmacia Biotechnology, Buckinghamshire, UK) in 1% skim milk in TBS-T for 1 h at room temperature. The targeted protein signals were detected by incubation with ECLTM Western Blotting Detection Reagents (Bio-Rad, Hercules, CA, USA) and developed on X-ray film (FUJIFILM Medical Systems, Minato City, Tokyo, Japan). The intensity of Western blot bands and respective ratios were quantified using ImageJ (1.53j) (https://imagej.net/, accessed on 13 July 2021).

Immunohistochemistry was performed on 5 μm thick paraffin-embedded EDTA-decalcified knee sections. Heat induced epitope retrieval was performed using a Citrate-EDTA buffer (pH 6.2) for 10 minutes at 95°C. Sections were treated with 3% H₂O₂/methanol for 10 minutes to inactivate endogenous peroxidase, blocked in goat serum for 30 minutes, and incubated overnight at 4°C with primary antibodies against DOT1L (Abcam, ab64077, 6 μg/ml) or HIF1A (Abcam, ab82832, 10 μg/ml) or for 90 minutes with primary antibody against H3K79me2 (Abcam, ab3594, 1 μg/ml). Rabbit IgG (Santa Cruz, sc-2027) was used as negative control. Avidin-biotin complex amplification (Vectastain ABC kit, Vector Laboratories) was used, except for the immunohistochemical detection of H3K79me2. Peroxidase goat anti-rabbit IgG (Jackson Immunoresearch) was applied for 30 minutes, and peroxidase activity was determined using DAB. Images were taken using an Olympus IX83 microscope. Quantification of the DAB staining was performed with a color deconvolution plugin (Jacqui Ross, Auckland University) in ImageJ Software (NIH Image). Quantification was performed using the average of 2 technical replicates for 5 different mice per condition, with staining intensity reported relative to the average of the 5 SHAM+Vehicle mice.

Sections were air-dried overnight at room temperature, followed by rehydration with phosphate-buffered saline (PBS; Sigma). The sections were incubated for 1.5 h in a blocking buffer containing 10% normal goat serum (Vector laboratories, Newark, CA, USA) diluted in PBST (PBS with 0.1% Triton X-100). For immunofluorescent labelling, the slides were incubated overnight at 4 °C in related primary antibodies (rabbit-anti-IBA1 1:200, 019-19741, WAKO; mouse-anti-ED1 1:100, MCA341GA, Bio-Rad; rabbit-anti-HIF1a 1:100, ab82832, Abcam; rabbit-anti-pimonidazole 1:200, HP3-1000, HPI Inc.; rabbit-anti-GLUT1 1:100, ab14683, Abcam; rabbit-anti-iNOS 1:100, PA1-036, Invitrogen; mouse-anti-3NT 1:100, ab61392, Abcam) diluted in a blocking buffer and then for 1 h in corresponding secondary antibodies diluted 1:200 (AlexFluor546-conjugated goat-anti-rabbit IgG, A11035; AlexFluor488-conjugated goat-anti-mouse IgG, A11001; Invitrogen), before being mounted with a DAPI-containing mounting medium (Biotium, Fremont, CA, USA). For labelling by immunohistochemistry, biotin-conjugated secondary antibodies (goat-anti-rabbit, BA1000, Vector laboratories) were used, and the slides were incubated in a biotin-streptavidin amplification solution (ABC kit; Vector laboratories) for 1 h, followed by a diaminobenzidine (DAB; Vector laboratories) reaction.

For HIF-1α studies cytosolic and nuclear fractions were isolated as described previously [13 (link)]. SDS-PAGE and blotting analysis were done using the following antibodies: HIF-1α (rabbit polyclonal against HIF-1α, ab82832 Abcam), TATA binding protein (TBP) (mouse monoclonal against TBP, ab51841, Abcam), and GAPDH (mouse monoclonal to GAPDH, ab8245, Abcam). Appropriate horseradish-peroxidase-coupled secondary antibodies were used, and chemiluminescence was performed with ECL-Prime (Amersham). Western blot replicates were scanned and quantified using ImageJ computer-assisted densitometric analysis. HIF-1α protein expression was normalized to GAPDH (cytosol) and TBP (nuclear compartment).

Protein expressions were determined by Western blot analysis as described in detail previously [16 (link)]. Primary antibodies for VEGF-A (512902, BioLegend, CA, USA) and HIF-1α (ab82832, Abcam, MA, USA) were added and incubated. All blots were incubated with anti-β-actin antibody (4970S, Cell Signaling Technology, MA, US) to confirm protein loading levels. Quantification of immunoblots was carried out using ImageJ.

The protein expression of HIF-1α in PC3 cells was assessed by Western blotting. Cells from 80% confluent cultures were washed with PBS and resuspended in lysis buffer for 20 min at 4°C, pelleted at 10000 rpm for 10 min. Protein (50 μg) from each electrophoresis was added into 5× SDS buffer and was boiled to denaturation. Stacking gel (80 V) and 120 V of separating gel were used to separate proteins, which then were transferred on to PVDF membranes with 300 mA for 1 h. Low-fat milk (5%) was used to block the membranes for 12 h at 4°C, and then the membranes were probed for 8 h at 4°C with primary rabbit anti-human antibody against HIF-1α (ab82832, Abcam, U.K.) and goat anti-rabbit horseradish peroxidase–conjugated secondary antibodies (ab205718, Abcam, U.K.) for 2 h at room temperature. Peroxidase labeling was revealed by ECL Western blotting detection system (Thermo, U.S.A.).