RNA immunoprecipitation and RNA pulldown

For RNA immunoprecipitation, lysates of MDA-MB-231 cells after hypoxia for 24hs were immunoprecipitated using anti-PKM2 and anti-PHD3 primary antibody. RNA immunoprecipitation was performed using Magna RNA immunoprecipitation RNA-Binding Protein Immunoprecipitation Kit (17–700, Millipore, Billerica, MA) following the manufacturer’s protocol.

For RNA pull-down, transcriptAid T7 High Yield Transcription Kit (Invitrogen, USA) was used to transcribe the biotin-labeled RNAs. Bio-16-UTP was added in the in vitro transcription. Briefly, 5 pmol of bio-labeled RNA was heated in RNA folded structure buffer (0.1 M KCl, pH 7, 10 mM MgCl₂, 10 mM Tris) for two minutes at 95 °C, then on ice for three minutes and then put for thirty minutes at room temperature. Folded RNA (5 μg) was then blended with cell lysates (5 mg) in 500ul Pierce™ IP Lysis Buffer (87787, Thermo Fisher Scientific) and incubated for 1 hr at room temperature. Dynabeads M-280 Streptavidin magnetic beads (50 μl, invitrogen, USA) were added into the binding reaction sample and further suspended for one hour. Washed beads were boiled in 1X protein loading buffer. The retrieved proteins were separated and analyzed by Western blot.

Mass spectrum followed with RNA pull down was performed to identify the proteins interacting with HIFAL. Three micrograms of bio-labeled RNA were heated for two minutes at 95 °C, then on ice for three minutes, provided with RNA folded structure buffer (0.1 M KCl, pH 7, 10 mM MgCl₂, 10 mM Tris) and transferred to at RT for thirty minutes in order to form normal secondary structure. 1 mg of cell lysis in RIP buffer were mixed with folded RNA and suspended at room temperature for 1 h. Then 60 microliters Streptavidin agarose beads (Invitrogen) were added into the interacting reaction sample and mixed for 1 h at room temperature. The beads were washed rotationally five times by Handee spin columns (Thermo), and then boilded in protein loading buffer. The pull-down protein was analyzed by western blot⁵³. Silver staining was conducted in terms of the manufacturer’s protocols with silver staining kit (LC6100, Thermo Fisher Scientific).