Illustra microspin columns

Immunoprecipitation and imunoblotting were performed as described previously [26 (link)]. The reagents were as following: anti-β-catenin (BD Bioscience, Cat# 610153), anti-γ-catenin (BD Bioscience, Cat# 610253), anti-CBP (Santa Cruz, clone SC-369), anti-p300 (Santa Cruz, clone SC-584), anti-activated- β-catenin (Millipore, clone 8E7), anti-lamin A/C (Santa Cruz, SC-7293), NE-PER Nuclear extraction reagent (Pierce, Cat#78833), Protease inhibitor cocktail (Calbiochem, Cat#539137), Protein A-agarose (Roche, Cat#11134515001), Illustra microspin columns (GE Healthcare, Cat#27-3565-01), ECL Plus (GE Healthcare, Cat#RPN 2132), and Blue ultra autorad film (BioExpress, Cat# F9029-8X10).

sRNAs were isolated from THP-1 cells using the mirVana kit (Ambion) according to the manufacturer’s instructions, precipitated with isopropanol and re-suspended in 10 µl RNase-DNase free water (Gibco), mixed with equal volume of gel loading dye (Ambion), heat denatured for 5 min and then cooled immediately on ice before separation on a 15% (w/v) denaturing acrylamide gel (1×TBE, 7 M urea, 15% acrylamide (29∶1 acryl:bis-acryl) in 1×TBE running buffer. The gel was stained with SYBR Gold (Invitrogen, Carlsbad, CA) for 5 min, and size fractions were collected by cutting the gel into three slices (<50 nt, 50–100 nt, 100–200 nt) according to a RNA molecular weight marker (RNA lowII, DynaMarker) (Figure S2 in File S1). The gel slices were transferred to a 2 ml clean tube RNase- and DNase-free and smashed by passing through a membrane-less filter (illustra MicroSpin columns, GE Healthcare). RNA was eluted in 1 ml of TEN buffer (Tris-HCl (pH 8.0) 8 mM, EDTA 0.1 M, NaCl 240 mM in RNase-free water), rocking overnight at 4°C. The tubes were then centrifuged 5 min at 10,000×g speed, the aqueous phase was recovered and the RNA was ethanol precipitated and re-suspended in RNase, DNase free water.