Fig. 1.

Barkor is a major component of the Beclin 1–PI3KC3 complex. (A) Silver staining of the tandem affinity-purified Beclin 1 complex or vector alone in U₂OS cells. All the marked bands were identified by mass spectrometry. (B) A similar Beclin 1 complex was purified from human kidney embryonic HEK293T cells. (C) Reciprocal coimmunoprecipitation of Barkor and Beclin 1. 293T cell extracts were immunoprecipitated with either anti-Barkor or Beclin 1 antibody and then analyzed. (D) Beclin 1 bridges the interaction between PI3KC3 and Barkor. Beclin 1-knockdown 293T cells or control cells were transfected with FLAG-PI3KC3 and Myc-Barkor. Whole-cell lysates were immunoprecipitated with anti-FLAG or Myc antibodies and analyzed. (E) Barkor-knockdown decreases the activity of PI3KC3 in vivo. Barkor-knockdown U₂OS cells were transfected with FYVE2-EGFP expression vector. Thirty hours after transfection, cells were treated with 5 mM 3-MA for another 4 h. FYVE2-EGFP was quantified in F.