Hypoxia-inducible factor 1-alpha (HIF1A) is a key transcription factor that orchestrates cellular responses to hypoxia. Under normal oxygen conditions, it undergoes rapid proteasomal degradation mediated by the von Hippel-Lindau (VHL) ubiquitination pathway. In hypoxia, however, HIF1A stabilizes and translocates to the nucleus, where it forms a heterodimer with HIF1β and binds to hypoxia-response elements (HREs), activating genes involved in angiogenesis, glycolysis, and erythropoiesis. Its dysregulation is implicated in cancer, ischemic disorders, and metabolic diseases, highlighting its importance as a therapeutic target. Notably, HIF1A directly interacts with E1A Binding Protein P300 (EP300) through its C-terminal transactivation domain, recruiting this coactivator to enhance histone acetylation and drive robust expression of hypoxia-inducible genes.To detect the activity of recombinant HIF1A , a functional ELISA assay was performed to evaluate the interaction between recombinant mouse HIF1A and recombinant mouse EP300. Briefly, HIF1A were diluted serially in PBS, with 0.01% BSA (pH 7.4). Duplicate samples of 100ul were then transferred to EP300-coated microtiter wells and incubated for 2h at 37℃. Wells were washed with PBST and incubated for 1h with anti-HIF1A pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody, wells were aspirated and washed 3 times. With the addition of substrate solution , wells were incubated 15-25 minutes at 37℃. Finally, add 50µL stop solution to the wells and read at 450nm immediately. The binding activity of HIF1A and EP300 was shown in Figure 1, and this effect was in a dose dependent manner.