Mini edta free

Cells (1x10⁶ cells were plated in matrigel-coated Primaria 60-mm dishes (BD Falcon) as described above. miRNAs were transfected by a standard forward transfection protocol. Seventy-two hr after transfection, cells were washed with ice-cold PBS and harvested in 80 μl of hypotonic buffer (50 mM Tris pH7.5, 10 mM NaCl, 3 mM MgCl₂, 10% glycerol) supplemented with protease inhibitors (complete tablets, Mini EDTA-free, Roche) and phosphatase inhibitors (cocktail 3 Sigma, 2mM orthovanadate, 1 mM NaF). After 1 min, cells were scraped and NP40 was added at 0.1% final concentration. After 5 min on ice, cells were centrifuged at 4000 rpm for 5 min at 4°C; the supernatant and cytosolic fractions were then recovered. Pellets were resuspended in 70 μl IPLS buffer (50 mM Tris pH 7.5, 120 mM NaCl, 1 mM EDTA, 0.5% NP40, supplemented with the same inhibitors as above) and sonicated with 3 pulses of 10 min each (30 s on/30sec off) on ice in a Bioruptor Plus (Biosense). Nuclear lysates were centrifuged at 16,000 rpm for 20 min at 4°C and the supernatant was recovered as nuclear fraction. Concentration of cytoplasmic and nuclear lysates were measured using the Bradford assay (Protein Assay Dye Reagent Concentrate, Bio Rad). Lysates (10 to 20 μg) were loaded for protein separation and subsequent blotting onto a PVDF membrane (Amersham Hybond PVDF, GE Healthcare).

Cells were lysed using standard RIPA lysis buffer (150 mM NaCl, 1% NP40, 1% (w/v) sodium deoxycholate, 0.1% SDS, 50 mM Tris pH 8) containing protease inhibitors (Roche mini EDTA-free, 1 tablet in 10 ml) and phosphatase inhibitors (2 mM Sodium Ortho-Vanadate, Roche Phosstop 1 tablet in 10 ml, 0.1 M sodium fluoride, 0.1 M beta-glycerophosphate) and benzonase (50 U/ml), after washing briefly with FBS-free DMEM. Lysates were clarified and protein concentration was determined using a BCA assay. Equal protein amounts were run on SDS-PAGE gels and transferred to a nitrocellulose membrane with 0.2 μm pore size. After Ponceau staining, membranes were incubated in 5% skim milk in PBST (134 mM NaCl, 2,7 mM KCl, 5.4 mM Na₂HPO₄, 1.47 mM KH₂PO₄) for 1 h, briefly rinsed with PBST and then incubated in primary antibody solution (5% BSA PBST or 5% skim milk PBST) overnight at 4°C. Membranes were then washed three times 15 min each in PBST, incubated in secondary antibody solution (1:10 000 in 5% skim milk PBST) for 1 h at room temperature, then washed again three times for 15 min. Finally, chemiluminescence was detected using ECL reagents and the Biorad ChemiDoc Imaging System. No membranes were stripped. Antibodies used in this study are listed in Supplementary Table S1.

For CoIP, protein was extracted by Pierce IP Lysis Buffer (87787, Thermo Fisher Scientific) with cOmplete Tablets, Mini EDTA-free, EASYpack (04693159001, Roche, Basel, Switzerland), and PhosSTOP EASYpack (04906837001, Roche). Briefly, the lysates were incubated with certain antibodies at 4°C overnight. Then, lysates were incubated with Pierce Protein A Magnetic Beads (88846, Thermo Fisher Scientific) for an hour at room temperature. After being washed by lysis buffer, beads were heated with SDS loading and analyzed by WB. Antibodies used in this study were listed in table S6.