RAW264.7 cells were transfected with miR-124 mimic or All Star negative control (NC) siRNA and cultured as described above for 24 h with RANKL. Then, cells were lysed and total cellular proteins were extracted using RIPA buffer (Cell Signaling Inc., Beverly, MA, USA). Western blot was performed as previously reported [4 (link)]. Membranes were incubated overnight at 4 °C with the following antibodies: NFATc1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 1:1000 dilution; p-ERK1/2 and ERK1/2 (Cell Signaling Inc., Beverly, MA, USA) at 1:1000; and β-actin (Sigma Aldrich, St. Louis, MO, USA) at 1:5000. The secondary antibodies Alexa Fluor 680 Goat anti-Rabbit (1:2000) and Alexa Fluor 800 Rabbit anti-Mouse (1:5000) (Molecular Probes, Life Technologies, Carlsbad, CA, USA) were incubated for 1 h at room temperature. Protein visualization and quantization were carried out with the LI-COR Odyssey scanner and software (LI-COR Biosciences, Lincoln, NE, USA). Blots were imaged using an Odyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) according to the manufacturer’s instructions. Experiments were repeated two times.
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