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. Author manuscript; available in PMC: 2016 Oct 2.
Published in final edited form as: Biochem Biophys Res Commun. 2015 Aug 25;465(4):739–745. doi: 10.1016/j.bbrc.2015.08.070

Figure 3. Beclin1 interacts with the components of the IKK complex.

Figure 3

(A) Flag-Beclin1 was co-transfected with GST or with GST-tagged IKKα, IKKβ or IKKγ in HEK293 cells. The interaction of these proteins was examined with GST pulldown, followed by anti-Flag immunoblot to detect Flag-Beclin1 (top panel). Protein inputs were shown in the middle and bottom panels. (B) PI3KC3-Flag was co-transfected with GST or with GST-tagged IKKα, IKKβ, IKKγ or Beclin1 in HEK293 cells, followed by GST pulldown assay and anti-Flag immunoblot. (C) The knockdown efficiency of the endogenous Beclin1 with lentiviral transduction of Beclin1-specific shRNAs in HEK293 cells was determined by anti-Beclin1 immunoblot. (D) The interaction of PI3KC3 with IKKα or IKKβ was examined in transfected NS- or Beclin1- depleted HEK293 cells as shown in the figure. (E) Schematic structures of the Beclin1 mutants, which were generated using PCR-based mutagenesis method. (F) and (G) The IKK-binding motifs on Beclin1 were evaluated using Flag-Beclin1 and its mutants, which were co-transfected with GST-tagged IKKα or IKKβ in HEK293 cells. (H) The knockdown efficiency of endogenous IKKα or IKKβ in HEK293 cells was determined by immunoblot with relevant antibodies. (I) The interaction of Beclin1 with IKKα or IKKβ was examined in transfected NS-, IKKα- or IKKβ-depleted HEK293 cells using the method as above.