Western Blot Analysis of Hypoxia Proteins

Preparation of whole cell lysates and nuclear extracts, gel electrophoresis and electroblotting were performed as previously reported (Sasagawa et al., 2018 (link)). The following primary antibodies were used: anti-HIF-1α (1:500, #3716; Cell Signaling Technology, Beverly, MA, USA), anti-HIF-2α (1:1000, NB100-122; Novus Biologicals, Littleton, CO, USA), anti-HIF-1β (1:2000, NB100-110; Novus Biologicals), anti-β-actin (1:1000, #4967; Cell Signaling Technology), anti-TATA binding protein (1:200, sc-421; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-human FLT1 N-terminal region (rabbit polyclonal) antibody (1:1000) (Tanaka et al., 1997 (link); Sasagawa et al., 2018 (link); Sasagawa et al., 2020 (link)).