Western Blot and miRNA Analysis in RMS

Cells or tissues were collected and lysed in TNES buffer (10 mm Tris, pH 8.0; 150 mm NaCl; 2 mm EDTA; 0.5% SDS). Equal 25 μg aliquots of proteins were electrophoresed on 8% SDS-polyacrylamide gels and electrotransferred onto polyvinylidine difluoride membranes. The blots were blocked with Tris-buffered saline containing 5% non-fat milk for 1 h. The membranes were incubated with specific antibodies: 1 : 1000 anti-TGF-β1, anti-Smad7, anti-SPRY4, anti-PKCα, anti-ERK, anti-JNK, and anti-p38MAPK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); and 1 : 1000 anti-p-ERK, anti-p-JNK, and anti-P-p38MAPK (New England BioLabs, Beverly, MA, USA) for 3 h at room temperature, all of which recognize the activated forms of these kinases. Proteins were visualized with peroxidase-conjugated secondary antibodies at 1 : 2000 for 1 h, using an enhanced chemiluminescence detection system (Santa Cruz). Total RNA was collected from RMS cell lines and frozen tissues according to the manufacturer's recommendations. Mature miRNA analysis was performed using TaqMan miRNA assays (Takara Shuzo, Kyoto, Japan), including RT and real-time PCR. RT reactions were performed using a single miRNA-specific stem-loop RT primer as described by Chen et al.^{44 (link), 45 (link)} (Supplementary Table1).