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. 2008 Dec 2;105(49):19211–19216. doi: 10.1073/pnas.0810452105

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© 2008 by The National Academy of Sciences of the USA

Freely available online through the PNAS open access option.

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Fig. 4. — Barkor and UVRAG form distinct subcomplexes with Beclin 1 through their CCDs. (A) Barkor binds to Beclin 1 through its CCD. 293T cells were transfected with FLAG-Beclin 1, Myc-Barkor, or its Myc-tagged mutants. Whole-cell lysates (WCLs) were immunoprecipitated (IP) with anti-Myc followed by immunoblotting (IB) with anti-FLAG. § indicates a nonspecific band. (B) Beclin 1 binds to Barkor through its CCD. 293T cells were transfected with Myc-Barkor, FLAG-Beclin 1, or its FLAG-tagged mutants. WCLs were immunoprecipitated with anti-FLAG followed by the IB with anti-Myc. (C) Direct interaction of Beclin 1 with Barkor or UVRAG. A Ni-column was incubated first with His-Beclin 1-CC and then with FLAG-tagged Beclin 1-CC, Barkor-CC, and UVRAG-CC. Proteins bound to beads and inputs were analyzed. (D) Barkor and UVRAG form distinct subcomplexes with Beclin 1. 293T cells were transfected with FLAG-UVRAG, Myc-Barkor, and HA-Beclin 1. WCLs were immunoprecipitated with anti-FLAG, anti-HA, or Myc, and the immunoprecipitates were analyzed. (E) Barkor competes with UVRAG for binding to Beclin 1. UVRAG was first incubated with Beclin 1 in vitro; increasing doses of the Barkor CCD were then added to the reactions. After extensive washing, the proteins bound to beads were analyzed by SDS/PAGE stained with Coomassie blue. (F) UVRAG competes with Barkor–Beclin 1 interaction in vivo. HA-Beclin 1 was cotransfected into HEK293T cells with Myc-Barkor or FLAG-tagged UVRAG. WCLs were immunoprecipitated with anti-HA followed by the IB with antibodies against Myc, FLAG, or HA.