Upregulation of Notch signaling components in rhabdomyosarcoma (RMS) cell lines. (a, left) Western blotting of Notch1-3 intracellular cleaved domains (IC), Jagged1, HES1 and β-actin (loading control) in whole-cell lysates from embryonal (ERMS) and alveolar (ARMS) RMS cell lines and normal human myoblasts SkMCs as control. Arrows indicate the IC domains of Notch1-3. Notch1IC was detected using the antibody that recognizes only the activated form (cleaved at Val1744). (a, right) Histograms report densitometric analysis of Notch1IC (Val1744), Notch2IC, Notch3IC, HES1, and Jagged1 bands normalized to β-actin of three independent experiments. (b) Western blot analysis of nuclear (N) and cytoplasmic (C) -enriched cell fractions of embryonal (ERMS) and alveolar (ARMS) RMS cell lines. Notch1IC (Val1744), Notch2IC and Notch3IC forms were detected in all cell lines. β-actin and topoisomerase IIβ were used as loading controls to discriminate the different cell fractions. (c) mRNA levels of Notch1-3, HES1 and Jagged1 (real time RT-PCR) were normalized to β-actin levels and expressed as fold increase over control SkMC (black column; 1 arbitrary unit). Columns, means; Bars, S.D. Results from three independent experiments are shown