Streptavidin magnetic beads

VSMC were seeded at 3.5x10⁵cells in 60 mm petri dishes in a 3 mL DMEM containing 10%serum fetal bovine for 24 h. Treatments, as indicated in each legend, were performed in serum-free medium. Biotinylation experiments with EZ-Link sulfo-NHS(N-hydroxysuccinimido)-biotin (Thermo-Scientific) for 1 h at 4 °C were performed as described [25] (link). The reaction was stopped with 50 mM Tris/HCl pH=7.5 for 10 min at 4 °C. After cell lysis (50 mM Tris-HCl, 1% Triton X-100, 150 mM NaCl) for 1 h at 4 °C, cells were incubated overnight with streptavidin magnetic beads (Promega), followed by pull- down, washing with PBS to remove proteins that were not biotinylated, incubation in sample buffer for 30 min, boiling for 5 min and then SDS-PAGE, followed by western blot. All western blots were performed with anti-PDIA1 RL90 from Thermo-Scientific.

Anti-MYC, Hemagglutinin tag, DHX36, DDX5, DDX17, DHX9, DHX29 and DHX30 antibodies were purchased from Abcam, the V5-tag antibody was obtained from Source BioScience, DDX3X antibody was ordered from Santa Cruz and FXR1 antibody was purchased from Cell Signaling. RNA oligonucleotides were ordered from Integrated DNA Technologies. Streptavidin magnetic beads were obtained from Promega and Strep-Tactin magnetic nanobeads were purchased from IBA.

MCF7 cells were seeded at 3.5 × 10[5] cells per 10-cm dish before treatment with either vehicle (PBS), BRACO 5 µg/mL (LD₁₅), or RHPS4 1.5 µM (LD₂₅) for 72h. Cells were then crosslinked using 1% formaldehyde/PBS for 5 min at 25 °C and the crosslinking was then quenched with 0.125 M glycine for 5 min. Cells were scraped and resuspended in G4RP buffer (150 mM KCl, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP40, RNase inhibitor (Roche), homebrew protease inhibitor cocktail). Cells were then sonicated using a Covaris m220 Ultrasonicator using default settings at 10% duty for 2 min. The sonicated fractions were then incubated with 100 µM BioTASQ (or 100 µM biotin for negative controls) overnight at 4 °C. Five percent of the sonicate was collected as input control. Ten micrograms of streptavidin-magnetic beads (Promega) was added and the extract was incubated for 2 h at 4 °C. Magnetic beads were then washed four times in G4RP buffer for 5 min. The beads were then incubated at 70 °C for 1 h to reverse crosslink. TRIZOL was then used to extract the RNA from the beads using the manufacturer’s instructions.