Sab4200085

To detect the effect of SiAgo2 transfection, western blotting was performed. Briefly, BMSCs were collected and lysed to obtain protein. The protein concentration was determined by a BCA protein assay (Thermo Fisher, USA). Twenty milligrams of total protein samples were separated on 4–12% Bis-Tris protein gels at 150 V for 40 min, transferred to a polyvinylidene fluoride membrane, and blocked in 10% western blotting buffer for 30 min. The membrane was then stained with a primary anti-Ago2 antibody (Sigma-Aldrich, SAB4200085, St. Louis, USA) for 1 h, washed with TBST 3 times for 5 min each, and stained for 1 h with secondary antibody before a final wash and detection with Femto ECL. ImageJ software (NIH, Bethesda, Maryland, USA) was used to analyze the densities of the protein bands.

Cells were plated and treated with DOX at a concentration of 0.2 µg/mL or equivalent volume of vehicle for 24 h. Cells were washed in cold PBS, scraped, and then lysed with a buffer containing 0.5% Nonidet, 0.5 mM DTT, 20 mM Tris-HCL pH7.5, 150 mM KCL, 2 mM EDTA, 1 mM NaF, and inhibitors of RNases, proteases, and phosphatases. Ten percent of total lysate was removed and kept as the input samples and the remainder used for immunoprecipitation. Ten micrograms of anti-AGO2 (11A9, SAB4200085, Sigma-Aldrich) or anti-IgG (Sigma-Aldrich) antibodies were bound to sepharose beads in the presence of heparin. Precleared lysates were then incubated with the appropriate antibody-bound beads, and the immunoprecipitated proteins were then washed and incubated with DNase I in the presence of DNase buffer (Promega) followed by protease K (New England Biolabs) in the presence of 2× protease buffer (New England Biolabs). RNA extraction was then performed using phenol chloroform separation and ethanol/sodium acetate precipitation. RNA pellets were washed in ethanol and quantified using a Nanodrop.

For sample preparation, cells were lysed in RIPA buffer (R0278, Sigma-Aldrich) mixed with protease inhibitor cocktail (1:25, 118735, Sigma-Aldrich) for 45 min on ice. Cells were then centrifuged at 10,000 × g for 10 min at 4°C. The supernatants were collected and transferred to a fresh tube. Samples were boiled at 95°C for 5 min in Laemmli buffer (1610737, BioRad). Proteins were separated on a 4–12 % SDS/PAGE gel. Gels were then transferred using the Transblot-Turbo Transfer system (BioRad). After transfer, membranes were blocked for 1 h in Tris-buffered saline (T5912, Sigma-Aldrich) with 0.1 % Tween20 (TBST, P1379, Sigma-Aldrich) and 5 % (wt/vol) non-fat dry milk (70166, Sigma-Aldrich) followed by overnight incubation at 4°C. The primary antibody was mouse anti-AGO2 (1:1,000; SAB4200085, Sigma-Aldrich). After two TBST washing steps of 15 min, membranes were incubated for 1 h at room temperature with HRP-conjugated secondary antibodies: anti-mouse (1:5,000; sc-2005, Santa Cruz Biotechnology). Actin staining was done using a monoclonal mouse anti-β-actin HRP (1:100,000; A3854, Sigma-Aldrich). Membranes were developed with the ECL Prime Western Blotting Detection Reagent (RPN2232, GE Healthcare). The signal was measured using a Chemidoc MP system (BioRad) and band intensities were quantified by densitometry using the ImageJ software.

Immunoprecipitation of miRNA was performed using monoclonal anti‐Ago1 (clone 4B8, SAB4200084, Sigma) and anti‐Ago2 (clone 11A9, SAB4200085, Sigma) produced in rat. Anti‐rat IgG (I4131, Sigma) was used in controls. Antibodies were treated with Pierce's EZ‐Link Sulfo‐NHS‐LC‐LC‐Biotin (#21338) to covalently tag the antibodies with biotin. To form AGO/anti‐AGO complexes, 100 μL of Streptavidin Mag Sepharose beads (GE28‐9857‐99) was combined with 12.5 μg of antibody and incubated for 30 minutes at room temperature, then washed twice with 1 mL of IP Lysis/Wash buffer. The mixture was combined with blood serum (1 mL per reaction), 3% IGEPAL^® CA‐630 (I8896, Sigma, final concentration), Protease Inhibitor Cocktail (PIC, P8340, Sigma) and RNasine (Takara, 2313A, 0.5 U/mL final concentration) and incubated for 1 hour at room temperature. Isolation of miRNA from Sepharose beads was performed using the miRNeasy kit (217004, Qiagen), after adding 200 μL of QIAzol Lysis Reagent.