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. 2018 Dec 11;10(12):507. doi: 10.3390/cancers10120507

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© 2018 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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ARRDC3 mediates NEDD4 dependent ITG β4 ubiquitination by serving as an adaptor molecule between NEDD4 and ITG β4. (A) Endogenous ITG β4 was immunoprecipitated in MDA-MB-231 cells transfected with HA-NEDD4, followed by IB with HA (left panel). MDA-MB-231 cells were transfected with HA-empty and HA-NEDD4. IP was performed with HA trap beads, followed by IB with ITG β4 (right panel). (B) NEDD4 knockdown MD-MB-231 cells by shRNA were co-transfected with HA-Ub and either ITG β4-Myc or Myc-empty vector. Ubiquitination was analyzed by IP with Myc trap beads and IB with HA antibody. Input: whole cell lysate. (C) MDA-MB-231 cells were co-transfected with GFP or GFP-ARRDC3 and HA or HA-NEDD4. IP was performed with HA trap beads, followed by IB with ITG β4, HA and GFP antibodies. (D) MCF-7 cells were transfected with siRNA negative control (NC) and siRNA NEDD4. After 48 h, the knockdown was evaluated by Western blotting with NEDD4 and β-Actin antibodies (Top). Immunofluorescence-based ITG β4 recycling assay were performed in NEDD4 knockdown MCF-7 cells as described in materials and methods. Immunofluorescence signals of ITG β4 (green) were captured by a fluorescence microscope with DIC optic (bottom). Scale bar: 100 μm. All experiments were repeated at least three times.

ARRDC3 mediates NEDD4 dependent ITG β4 ubiquitination by serving as an adaptor molecule between NEDD4 and ITG β4. (A) Endogenous ITG β4 was immunoprecipitated in MDA-MB-231 cells transfected with HA-NEDD4, followed by IB with HA (left panel). MDA-MB-231 cells were transfected with HA-empty and HA-NEDD4. IP was performed with HA trap beads, followed by IB with ITG β4 (right panel). (B) NEDD4 knockdown MD-MB-231 cells by shRNA were co-transfected with HA-Ub and either ITG β4-Myc or Myc-empty vector. Ubiquitination was analyzed by IP with Myc trap beads and IB with HA antibody. Input: whole cell lysate. (C) MDA-MB-231 cells were co-transfected with GFP or GFP-ARRDC3 and HA or HA-NEDD4. IP was performed with HA trap beads, followed by IB with ITG β4, HA and GFP antibodies. (D) MCF-7 cells were transfected with siRNA negative control (NC) and siRNA NEDD4. After 48 h, the knockdown was evaluated by Western blotting with NEDD4 and β-Actin antibodies (Top). Immunofluorescence-based ITG β4 recycling assay were performed in NEDD4 knockdown MCF-7 cells as described in materials and methods. Immunofluorescence signals of ITG β4 (green) were captured by a fluorescence microscope with DIC optic (bottom). Scale bar: 100 μm. All experiments were repeated at least three times.