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. Author manuscript; available in PMC: 2014 Feb 6.
Published in final edited form as: Biochim Biophys Acta. 2012 Jun 15;1823(10):1686–1696. doi: 10.1016/j.bbamcr.2012.05.032

Fig. 1.

Fig. 1

Rad6B and β-catenin exhibit direct interactions. (A) Immunohistochemical analysis of Rad6 (a, b, c) and β-catenin (a′, b′, c′) in reduction mammoplasty (a, a′) and clinical breast carcinomas (b, b′, c, c′). (B) Immunofluorescence localization of Rad6 (a, b and c) and β-catenin (a′, b′ and c′) in reduction mammoplasty (a–a″), infiltrating ductal carcinoma (b–b″), and WS-15 breast cancer cells (c–c″). Magnification ×20 (a–a″ and b–b″) and ×100 (c–c″). (C) Co-immunoprecipitation analysis. Lane 1, 1/5th of MDA-MB-231 cytosol inputs. (D) Mapping β-catenin interacting regions. Pull-down assays from GSH-beads with His-tagged wild type Rad6B 1–152 (lanes 9–11) and GST-tagged β-catenin 1–781 (lane 9), β-catenin 131–422 (lane 10) or β-catenin 181–422 (lane 11) proteins. Lanes 1–4, inputs of His-Rad6B, GST-β-catenin 1–781, GST-β-catenin 131–422, and GST-β-catenin 181–422, respectively. Lanes 5–8, control GST, GST-β-catenin 1–781, GST-β-catenin 131–422, and GST-β-catenin 181–422, respectively. Blots were probed with GST (upper) and His (bottom) antibodies. (E) Mapping Rad6B interaction sites. Pull-down assays were performed with His-tagged Rad6B 1–152 (lanes 4 and 7), Rad6B 50–152 (lanes 5 and 8) or Rad6B 116–152 (lanes 6 and 9) and GST-β-catenin 1–781 (lanes 4–9) on GSH-beads (lanes 4–6) or Ni+ 2-beads (lanes 7–9) beads. Lanes 1–3, inputs of Rad6B 1–152, Rad6B 50–152, Rad6B 116–152, and GST-β-catenin 1–781. Blots were probed with His (upper) and GST (bottom) tag antibodies. Rad6B/β-catenin interaction regions are shown schematically in the right panels of D and E.