Ab51608

Standard western-blot assays were used to analyze protein expression in cells. The following antibodies were used for assays: anti-Flag-M2 (F1804, Sigma, 1:10000), anti-β-Actin (A5441, Sigma, 1:10000), anti-Myc (9E10, Santa Cruz, 1:1000), anti-HA (#2367, Cell Signaling, 1:1000), anti-Parkin (4211, Cell Signaling, 1:500), anti-HIF-1α (ab51608, Abcam, 1:500), anti-VHL (#2738, Cell Signaling, 1:500), anti-ubiquitin antibody (sc-8017, Santa Cruz,1:2000), anti-PINK1 (ab23707, Abcam, 1:1000), anti-GST antibody (sc-138, Santa Cruz, 1:5000), and anti-His antibody (sc-803, Santa Cruz, 1:1000). The band intensity was quantified using Image J software (NIH, Bethesda). Uncropped scans of western blots presented in the main figures are provided in Supplementary Figs. 10-15.

Immunostaining protocol was used as comprehensively described elsewhere [54 (link)]. Briefly, fixed cells were incubated with primary antibodies anti-HIF-1α (ab51608, 1:500) and anti-LC3B (ab52862, 1:500) (Abcam, Cambridge, UK) at 4°C overnight and secondary antibody conjugated to Texas Red (1:1000, T2767) (Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. Nuclei were visualized using DAPI staining and F-actin was detected using phalloidin staining. Digital cell images were captured using an Olympus BX61 fluorescence microscope equipped with a DP72 CCD camera and Olympus CellF software.

CHIP assay was employed to verify whether specific transcription factor and genomic DNA segment were colocalized into the same complex, in accordance with manufacturer’s instructions of the CHIP kit (P2078, Beyotime Biotechnology, China). After treating with 1% formaldehyde and Glycine solution, the cells were collected, lysed, and incubated with either the anti-HIF-1α antibody (ab51608, Abcam, Cambridge, UK) or the control IgG antibody (PP64B, Millipore, Bedford, MA, USA) at 4 °C, and shaken overnight. After incubating with 5 M NaCl at 65 °C for 4 h and proteinase K at 45 °C for 60 min, the enriched ITGB2 gene promoter fragment was purified and extracted for qRT-PCR analysis.

Total proteins were extracted from cells or tissues using RIPA lysis buffer (Promega, Madison, WI, USA). Proteins were separated on an 8% SDS-PAGE gel and then transferred to PVDF membranes. The membrane was blocked with 5% skim milk at room temperature for 1 h and then incubated with primary antibodies (anti-DLAT, 1:1000 dilution, Abcam, ab51608; anti-SP1, ab101562, 1:1000 dilution, Abcam) at 4 °C overnight, followed by incubating with horseradish peroxidase (HRP) conjugated β-actin secondary antibodies (1:5000) at room temperature for 90 min. The protein bands were scanned and visualized by the GS700 imaging densitometer (Bio-Rad Laboratories) and analyzed by Image Studio software.

Tissues were immediately fixed in 10% neutral formalin solution for 48–72 h after surgery. The dehydration instrument completes the graded dehydration and immerses the tissues in wax. The 4 μm sections were baked in a 67 °C oven for more than 4 h after the paraffin embedding, and then deparaffinized in xylene and graded ethanol in distilled water. Immunohistochemistry uses a two-step method. Antigen retrieval was performed with a microwave in a water bath with Tris-EDTA solution for 2 * 8 min. Then, endogenous peroxides were blocked in 3% H2O2 for 15 min at room temperature, Subsequently, the corresponding primary antibody working solution was incubated at 4 °C overnight, and the secondary antibody, goat anti-rabbit IgG (Dako, Shanghai, China) is incubated at 37 °C for 65 min, the sections were incubated with DAB working solution for 3 min. Finally, hematoxylin was used to counterstain the cell nuclei, and the sections were dehydrated, cleared and fixed with neutral gum. The immunohistochemical pictures were collected by an Olympus IX83 microscope. The antibodies used in this study were: c-Jun (Abcam, ab32137, rabbit), c-Fos (Abcam, ab222699, rabbit), HIF-1 (Abcam, ab51608, rabbit).

Western blot was performed in cultured cells following various treatments. The protein lysates (1% NP40, 50 mM Tris, 5 mM EDTA, 1% SDS, 1% sodium deoxycholate, 1% Triton X-100, 1 mM PMSF, 10 mg/ml aprotinin, 1 mg/ml leupeptin, and pH = 7.5) were measured by Bradford assay and the same amount of proteins was resolved on SDS-PAGE followed by an electric transfer to a PVDF membrane. The blots were blocked by 5% non-fat milk and incubated with primary antibodies, including HIF-1α (ab51608, 1:1000; Abcam, Cambridge, MA, United States), VEGF (ab51745, 1:2000; Abcam, United States) and GAPDH (ab9484, 1:1000; Abcam, United States). The blots were then incubated by appropriate HRP conjugated secondary antibodies, and signals were visualized by an enhanced ECL-based imaging system.

HUVECs were lysed and subjected to western blot analysis as described. Total protein form HUVECs was collected by lysis buffer and then separated by 10% SDS-PAGE gel. The isolation was transferred onto the PVDF membrane. The membranes were incubated with primary and secondary antibodies. Antibodies used in the blot were listed as follows: anti-HIF1α (Abcam, ab51608, 1:1000), anti-VEGFA (Abcam, ab52917, 1:1000). The blot signal detection was analyzed using chemiluminescent reagent.