Protease k

For RNP-IP, the Dynabeads^® Protein G (Thermo Fisher Scientific, Rockford, IL, USA) were coated with control IgG (Santa Cruz Biotechnologies, Santa Cruz, CA, USA) or Ago2 antibody (Sigma, St. Louis, MO, USA). Cytoplasmic lysates were prepared using the protein extraction buffer (PEB) containing protease/phosphatase inhibitors and RNaseOUT (Invitrogen, Carlsbad, CA, USA). Equal amounts of lysates were incubated with antibody-coated Dynabeads for 4 h at 4 °C. After washing several times with PEB buffer, the Ago2-IP materials were treated with DNase I (Ambion, Austin, TX, USA) and protease K (Bioneer, Daejeon, South Korea). Proteins were denatured with acid phenol (Ambion) and RNAs were precipitated with absolute ethanol overnight at −20 °C. The level of mRNA in Ago2-mediated RNA-induced silencing complex (RISC) was determined by RT-qPCR as described above.^{19 (link)}

The cross-section morphology of the scaffolds was observed using scanning electron microscopy (SEM; GENESIS-1000, Emcraft, Gwangju, Korea). The thermal property of the scaffolds was analyzed by a thermal gravimetric analyzer (TGA 4000, PerkinElmer, Waltham, MA, USA). To assess the neutralization capacity of the scaffolds, the mass and pH changes were measured in 500 μL phosphate-buffered saline (PBS) solution (pH 7.4) with 20 µg/mL protease K (Bioneer, Daejeon, Korea) for 14 days. The inorganic compositions of the scaffold were measured using inductively coupled plasma-optical emission spectroscopy (ICP-OES, Optima 8000, PerkinElmer, Waltham, MA, USA). The water contact angle (WCA) was analyzed using the sessile drop method at room temperature to evaluate the hydrophilicity and hydrophobicity of the scaffolds.