Molecular Interactions of HIF-1α

Extraction of the total cell or nuclear lysates and western blot analysis were performed as reported previously [18 (link)]. Reverse transcription PCR using published primer sequences and immunoprecipitation were also performed as described previously [19 (link)]. [³⁵S]Methionine-labeled in vitro translated HIF-1α was prepared using the TNT system (Promega, WI, USA). GST-fusion proteins were expressed in E. coli BL21 (DE3) and induced with 0.4 mM isopropyl-thio-β-D-galactopyranoside. Equal amounts (1 μg) of GST and GST-MTA1 immobilized on glutathione sepharose beads were incubated with in vitro translated HIF-1α in modified GBT buffer (10% glycerol, 50 mM Hepes-NaOH [pH 7.5], 170 mM KCl, 7.5 mM MgCl₂, 0.1 mM EDTA, 0.1 mM DTT, and 1% Triton X-100) at 4°C for 3 h. After washing, the bound proteins were eluted with the sample buffer and were separated by SDS-PAGE, followed by western blot analysis.