Immun star horseradish peroxidase luminal enhancer

Frozen tissue from the 2 samples with VGLL2-CITED1 fusions (SRMS6, SPRMS7) and one control sample (normal skeletal muscle) were homogenized in Tissue Protein Extraction Reagent (Thermo Scientific, Cat# 78510) supplemented with protease inhibitors. Electrophoresis and immunoblotting were performed on the protein extracts using 30 μg of protein per sample. VGLL2 and β-Actin expression were detected by anti-VGLL2 mouse polyclonal antibody (Abcam, Cat# ab169247) with 1:500 dilution and anti-Actin mouse monoclonal antibody (Cell Signaling, Cat#3700) with 1:1,000 dilution, respectively. Following hybridization with the secondary rabbit anti-mouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, #sc-358923) with 1:5,000 dilution, the blots were incubated with Immun-Star horseradish peroxidase luminal/enhancer (Bio-Rad, Hercules, CA, USA) and exposed onto Kodak Biomax MR Film (Eastman Kodak Co., Rochester, NY, USA).

Frozen tissue from the 2 index AS cases and 10 control samples, including 5 CIC wild-type AS, 2 CIC-DUX4 positive small blue round cell tumors (SBRCTs), 3 gastrointestinal stromal tumors (GISTs), were homogenized with Tissue Protein Extraction Reagent (Thermo Scientific, Waltham, MA) supplemented with protease inhibitors. Electrophoresis and immunoblotting were performed with the polyclonal anti-CIC antibody (ABN446, 1:1,000, Millipore, Billerica, MA). A β-actin expression was used as reference with the monoclonal anti-actin antibody (8H10D10, 1:10,000, Cell Signaling, Danvers, MA). Following hybridization with the secondary antibodies, the blots were incubated with Immun-Star horseradish peroxidase luminal/enhancer (Bio-Rad, Hercules, CA) and exposed onto Kodak Biomax MR Film (Eastman Kodak, Rochester, NY).