Hif1α transcription factor assay kit

CD4⁺ T cells were activated with anti-CD3 (Clone#145-2C11, Cat#BE0001-1, Bio X Cell) and anti-CD28 mAb (Clone#37.51, Cat#BE0015-1, Bio X Cell) in the presence or absence of butyrate (0.5 mM) for 24 h. Cells were collected, and nucleoprotein was extracted by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Cat#78835, ThermoFisher), and HIF1α activity in samples was measured using HIF1α Transcription Factor Assay Kit (Cat#ab133104, Abcam). Nuclear protein was diluted with Complete Transcription Factor Binding Assay Buffer, and then incubated in the wells of Transcription Factor HIF1α plate overnight at 4 °C. Primary HIF1α antibody was then incubated in the wells for 1 h at room temperature, followed by the addition of the second goat anti-rabbit HRP antibody for another 1 h. Then, Transcription Factor Developing Solution was then incubated in the wells for 30 min at room temperature. After adding the Stop solution, HIF1α activity levels were measured at 450 nm using BioTek Gene5 instrument.

To determine the effect of MAP17 on HIF1α transcriptional activity, nuclear extract lysates were obtained from MAP17 knockdown or MAP17-expressing HCC cells by using a Nuclear Extraction Kit (#2900, Millipore). The activities of HIF1α in nuclear extract lysates were detected using the HIF1α Transcription Factor Assay Kit (ab133104, Abcam) according to the manufacturer’s protocols.

An HIF-1α Transcription Factor Assay Kit (Abcam) was used to evaluate DNA-binding activity of HIF-1α in the nuclear fraction of the heart primordium according to the manufacturer’s instructions. The nuclear fraction was obtained by using a Nuclear Extraction Kit (Abcam).