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. 2017 Sep 1;31(17):1754–1769. doi: 10.1101/gad.302497.117

Figure 5.

Figure 5.

Bim–DLC1 binding is required for efficient binding of BimEL to anti-apoptotic Bcl-2 proteins. (A) Binding of wild-type BimEL versus BimELAA to anti-apoptotic Bcl-2 proteins. Whole-cell lysates (1% Triton X-100) of Bax−/−Bak−/− MEFs overexpressing either murine V5-tagged BimEL or the DLC1-binding mutant V5-BimELAA were subjected to anti-V5 immunoprecipitation. Precipitated proteins were identified by Western blotting with the antibodies indicated. Data are representative of three independent experiments. (B) BimEL binding to anti-apoptotic proteins is reduced by RNAi against DLC1. Bax−/−Bak−/− MEFs overexpressing murine V5-tagged BimEL were transfected with control siRNA or siRNA against DLC1. After 48 h, whole-cell lysates (1% Triton X-100) were prepared and subjected to anti-V5 immunoprecipitation followed by Western blotting for the proteins indicated. Data are representative of three independent experiments. (C) In the presence of overexpressed wild-type BimEL but not BimELAA, Bcl-2 and Bcl-XL run in higher-MW complexes in BN-PAGE. Mitochondrial lysates (1% digitonin) from Bax−/−Bak−/− MEFs overexpressing either murine V5-tagged BimEL wild type or BimELAA were run on a 6%–16.5% BN-PAGE gradient gel and subjected to Western blotting with the antibodies indicated. VDAC was probed to test whether it may be contained in the complexes. Data are representative of at least three independent experiments. (D) Bim-induced high-MW Bcl-XL complexes are reduced upon RNAi against DLC1. Mitochondria were prepared from Bax−/−Bak−/− MEFs overexpressing murine V5-tagged BimEL 28 h after transfection with control or DLC1-specific siRNA. Complexes were separated on a 6%–16.5% gradient BN-PAGE gel and subjected to Western blotting with antibodies specific for Bcl-XL. Data are representative of three independent experiments. (E) Overexpression of wild-type BimEL, but not BimELAA, induces Bcl-XL high-MW complexes as assessed by gel filtration. Lysates of mitochondria from Bax−/−Bak−/− MEFs overexpressing murine V5-tagged BimEL wild type or BimELAA were separated by gel filtration on a Superdex 200 10/300 GL column. The same collected fractions were used as in Figure 2E. (F) Binding of Bcl-XL to Bim preferentially occurs in DLC1-induced Bim complexes. Recombinant proteins (BimL, DLC1, and Bcl-XL) were incubated with LUVs as in Figure 1I. Recombinant Bcl-XL (100 nM) was added as indicated, and Bim-containing complexes were detected by BN-PAGE and anti-Bim Western blotting. Data are representative of three independent experiments (two experiments with Bim + Bcl-XL only).