The western blot has been performed as described previously [17 (link)]. Primary antibodies against proteins were as follows: ATF3 (1:1000, Catalog number: ab207434, Abcam, UK), ZNF24 (1:1000, Catalog number: 11219-1-AP, Proteintech, Rosemont, USA), VEGFA (1:1000, Catalog number: 26157-1-AP, Proteintech, Rosemont, USA), HIF-1α (1:1000, Catalog number: 20960-1-AP, Proteintech, Rosemont, USA), HDAC1 (1:1000, Catalog number: 10197-1-AP, Proteintech, Rosemont, USA), and β-actin (1:1000, Catalog number: 20536-1-AP, Proteintech, Rosemont, USA).
The Co-IP assay has been performed as described previously [18 (link)]. Cells lysates were obtained by cell lysis buffer for IP (Beyotime). Take a small number of cell lysates for western blot analysis. IgG (Cell Signaling Technology) or primary antibodies: anti-HIF-1α (Proteintech, Rosemont, USA), anti-HDAC1 (Proteintech, Rosemont, USA) were added to the cell lysates, and incubated on a rotating platform at 4 °C overnight. Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added to the mixture and incubated with slow shaking at 4 °C for 2 h. Then separated the agarose, and analyzed the sample through western blot.
The Co-IP assay has been performed as described previously [18 (link)]. Cells lysates were obtained by cell lysis buffer for IP (Beyotime). Take a small number of cell lysates for western blot analysis. IgG (Cell Signaling Technology) or primary antibodies: anti-HIF-1α (Proteintech, Rosemont, USA), anti-HDAC1 (Proteintech, Rosemont, USA) were added to the cell lysates, and incubated on a rotating platform at 4 °C overnight. Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) was added to the mixture and incubated with slow shaking at 4 °C for 2 h. Then separated the agarose, and analyzed the sample through western blot.
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