Fxr1 monoclonal antibody

FXR1 RNP IP is performed as previously described [36 (link)] with some modifications. Briefly, cell lysates are prepared from exponentially growing UMSCC74B cells. Equal amounts of protein are used (750–1000μg). FXR1 monoclonal antibody (Millipore) or isotype control IgG (Santa Cruz) are pre-coated onto protein A/G Sepharose beads (PAS) and extensively washed using NT2 buffer [50mM Tris–HCl, 150mM NaCl, 1mM MgCl₂, 0.05% Nonidet P-40 (NP-40), pH 7.4]. Individual pull-down assays are performed at 4°C for 1–2 h to minimize potential reabsorbing of mRNAs. For RNA analysis, the beads are incubated with 1ml NT2 buffer containing 20 U RNase-free DNase I (15 min, 30°C), washed twice with 1ml NT2 buffer and further incubated in 1 ml NT2 buffer containing 0.1% SDS and 0.5mg/ml proteinase K (15 min, 55°C) to digest the proteins bound to the beads. RNA is extracted using phenol and chloroform, and precipitated in the presence of glycogen. For analysis of individual mRNAs, the RNA isolated from the IP is subjected to reverse transcription (RT) using random hexamers and SuperScriptII reverse transcriptase (Biorad).

FXR1 RNP IP is performed as previously described [67 (link)] with a few modifications. Briefly, an equal amount of total protein lysate is used (≥1mg) for each RIP. FXR1 monoclonal antibody (Millipore) or isotype control mouse IgG is pre-coated onto protein A/G plus agarose beads. Beads are extensively washed using NT2 buffer (50mM Tris–HCl, 150mM NaCl, 1mM MgCl₂, 0.05% Nonidet P-40 (NP-40), pH 7.4) supplemented with RNase inhibitors. Individual pull-down assays are performed at 4°C for 2–4 h to minimize potential reabsorbing of mRNAs. For RNA analysis, the beads are incubated with 1mL NET2 buffer (1mL: 850μL NT2 with 10μL 0.1M DTT, 30μL 0.5M EDTA, RNase inhibitors) containing 20 U RNase-free DNase I (15 min, 30°C), washed 3X with NT2 buffer and further incubated in 100μL NET2 buffer, 100μL proteinase K buffer (2X: 50mM Tris–HCl, 100mM NaCl, 20mM EDTA, 1% SDS, pH 7.4), and 0.5mg/mL proteinase K (30 min, 55°C) to digest the proteins bound to the beads. RNA is extracted using Tri-Reagent.