Qexactive lc ms ms

The Q-Exactive LC-MS/MS (Thermo Fisher) was used to analyze urolithin A using a C18 column [(Waters Ltd., MA, USA) ACQUITY UPLC^® BEH C18, 2.1 × 50 mm, 1.7 μm; 0.5 ml/min] and HR-ESI-MS scan range from 150.0 to 2,000.0 Da. Sheath gas flow rate, 45 arb; aux gas flow rate; 15 arb; Capillary temp:320°C; S-Lens RF Level 50 T. chromatographed using LC-MS/MS at 30°C. The mobile phase consisted of solvent A (water, 0.1% formic acid) and solvent B (acetonitrile). Separation was performed at a flow rate of 0.5 ml/min with the following gradient elution: 0–10.5 min, a linear gradient runs from 10 to 100% solvent B; 10.5–12.5 min, 100% solvent B; 12.5–12.6 min, a linear gradient runs from 100 to 10% solvent B; 12.6–16.5 min 10% solvent B for re-equilibration. The limits of detection (LODs) were determined based on a signal-to-noise ratio (S/N) of 3 and of 10 for the limit of quantification (LOQ). The LODs: 0.5 μM (EA and urolithin A), 0.2 μM (urolithin C), the dynamic range: 0.5–20 μM. Urolithin A and urolithin C in the fermentation broth were first identified through HPLC and further identified by comparing the molecular mass of the compound obtained with that of a pure standard by using LC-MS/MS (35 (link), 36 (link)).

RNA pull-down was performed as previously described
[19] (link). First,
lncRP11-675F6.3 and its antisense RNA were transcribed
in vitro using the pGEM-3Z vector and T7 RNA polymerase RNA production system (Promega, Madison, USA). RNA was purified with an RNeasy® Mini kit (Qiagen, Valencia, USA) and treated with DNase I (Quanta Biosciences, Beverly, USA). RNAs were labelled with biotin using T4 RNA ligase sense, and the labelled RNA was captured with streptavidin magnetic beads. HepG2 cells were lysed and the extracts were mixed with the magnetic beads. The binding proteins on the beads were eluted and subsequently separated through SDS-PAGE followed by silver staining (Thermo Scientific, Waltham, USA). The differential band was cut and then subjected to be analyzed by Q Exactive LC-MS/MS (Thermo Scientific). The candidate binding protein was further verified by western blot following RNA pull-down.
RNA immunoprecipitation (RIP) was performed according to the manufacturer’s protocol (Millipore). Briefly, cells were lysed and immunoprecipitated using an antibody against HK1 (1:50; Millipore) with protein A/G magnetic beads. The complexes bound to the magnetic beads were immobilized with a magnet, and the beads were washed to remove unbound materials. Finally, binding RNA was extracted and analyzed by real-time fluorescence quantitative PCR (RT-qPCR).