Anti hif 1α antibody

Total proteins from the treated retinal endothelial cells were extracted with RIPA lysis buffer (Beyotime; cat.: P0013C) containing PMSF (1:100; Beyotime; cat.: ST506). The protein concentration was quantified by BCA assay (Sangon biotech; cat.: C503061) according to the manufacturer’s instruction, and further adjusted to 2 mg/ml before dodecyl sulfate, sodium salt-polyacrylamide gel electrophoresis (SDS-PAGE). When the SDS-PAGE gel electrophoresis was completed, the proteins were transferred onto a nitrocellulose filter membrane (NC; Millipore; cat.: HATF00010). After blocking with 5% milk in PBST buffer for 1 h, the membrane were incubated with the primary antibodies anti-STAT3 (1:1000; Cell Signaling Technology; cat.: 9139S), anti-p-STAT3 (1:1000; Cell Signaling Technology; cat.: 9145 T), anti-HIF-1α antibody (1:1000; Proteintech; cat.: 20,960-1-AP), anti-VEGFA (1:1000; Abcam; cat.: ab1316) and anti-α-tubulin (1:8000; Abcam; cat.: ab18207) at 4°C overnight. After that, the membrane was incubated with the corresponding secondary antibody conjugated with HRP (1:1000; Beyotime; cat.: A0208) for 2 h at room temperature. After rinsing with PBST buffer for three times, the immunoreactivity of membrane was visualized using the SuperSignal West Pico kit (Pierce) [20 (link)].

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (D-MEM/F-12, Gibco, USA), bovine serum albumin (BSA), and trypsin/EDTA (Gibco-BRL); EGM-2 Bullet Kit (mixture of ascorbic acid, hydrocortisone, EGF, IGF-I, heparin, VEGF, and FGF2,; Lonza); insulin-transferrin-selenium sodium pyruvate (ITS; Invitrogen); Jagged1 Fc Chimera (599-JG; R&D Systems); rat immunoglobulin G (IgG) (I4131; Sigma Aldrich); PE-mouse anti-Rat CD31 (BD Pharmingen™); anti-Hif-1α antibody (Proteintech); R-PE-conjugated Donkey Anti-Goat IgG(H + L) (Proteintech); anti-Jagged-1 antibody (Affinity); anti-N1ICD antibody (CST); anti-Hey1 antibody (Abcam); anti-VEGF receptor2 antibody (Abcam); anti-Von Willebrand factor (vWF) antibody (Abcam); goat anti-rabbit IgG Alexa Fluor 594 (Invitrogen); TRIzol (Takara, Biotechnology, Dalian, China); 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes); reverse Q-PCR kit, and RT-PCR kit, protein lysis buffer kit (Beyotime Biotechnology); anti-actin antibody (Sigma); horseradish peroxidase (HRP)-conjugated goat anti-rabbit and goat anti-mouse IgG (Beyotime Biotechnology); Matrigel (Corning); Dual-GLO Luciferase Assay System (Promega); Fugen6 (Roche).

The ESCC and HEK 293 T cells were subjected to lysis using M-PER Mammalian Protein Extraction Kit lysis buffer (Thermo Scientific, 78501) for protein extraction. Co-immunoprecipitation (co-IP) of Flag-Pol ι, anti-Myc-USP7, or HA-HIF-1α proteins was performed using anti-Flag, anti-Myc, and anti-HA agarose beads (20 µl) for target precipitation, respectively. The endogenous level of HIF-1α was detected using the anti-HIF-1α antibody (Proteintech, 20960-1-AP, 1:100), while rabbit-purified IgG was employed as a negative control. Immunoprecipitation was carried out using protein G-conjugated magnetic beads (Abcam, ab193262) in accordance with the manufacturer’s instructions. Quantitative analysis of the western blotting was analyzed with Image J.