Mgcl2

Tyrosine phosphorylated peptides were custom synthesized by Sangon Biotech (Shanghai, China), the peptide sequence of Six2 was GEETSYCFKEKS, and the sequence of H2AX was GPKAPSGGKKATQASQRY. All peptides were purified by high-performance liquid chromatography, and all purified products were analyzed by mass spectrometry. The purity of each peptide was greater than 95%. Purified Eya1 protein (400 ng) was incubated in a final volume of 20 μL with 1 mM peptide in a buffer containing 60 mM HEPES (Beyotime Biotechnology, Shanghai, China), 75 mM NaCl (Beyotime Biotechnology, Shanghai, China), 75 mM KCl (Beyotime Biotechnology, Shanghai, China), 5 mM MgCl2 (Beyotime Biotechnology, Shanghai, China), 1 mM EDTA (Beyotime Biotechnology, Shanghai, China), and 1 mM DTT (Beyotime Biotechnology, Shanghai, China) at 37°C for 2 h. The released phosphate was quantified using the Malachite Green Phosphate Assay Kit (Sigma-Aldrich, St. Louis, USA). Duplicate values were background corrected and compared to the internal phosphate standard.

To visualize stable PFKFB3, γH2AX, ATM, MRE11, RAD50, and NBS1 foci at the DNA damage sites induced by H₂O₂ treatment, the HUVECs grown on the slides were treated with nuclear fraction procedures to remove soluble cytoplasmic proteins and loosely held nuclear proteins, as previously described [60 (link),70 ]. The HUVECs were first washed with PBS and then incubated for 3 min on ice in a cytoskeleton (CSK) buffer (pH 7.0) containing 10 mM PIPES (Sigma Aldrich), 100 mM NaCl (Sigma Aldrich), 300 mM sucrose, 3 mM MgCl₂ (Beyotime), 1 mM EGTA (Beyotime), and 0.7% Triton X-100. After the removal of the cytoplasm, the slides were washed three times with PBS and treated with the procedures of immunofluorescence microscopy.