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. Author manuscript; available in PMC: 2017 Nov 2.
Published in final edited form as: Cell Rep. 2017 Oct 10;21(2):442–454. doi: 10.1016/j.celrep.2017.09.052

Figure 4. ARTS binds to the BH3 domain of Bcl-2.

Figure 4

A. A custom designed Bcl-2 derived peptide array (CelluSpot™) was incubated with recombinant (His)6-Lipo-TEV (HLT-) tagged ARTS. The peptide array was also incubated with recombinant HLT-tag alone which served as a negative control. The arrays were probed with the indicated antibodies. Lower panel depicts the Bcl-2 peptides which bind to ARTS. These peptides are schematically represented across the Bcl-2 domains; the black lines indicate strong binding and the grey lines a weak to moderate binding. B. Bcl-2 expression vectors; full length and four constructs deleting each BH domain in Bcl-2 are illustrated at the top (FLD- Flexible Loop Domain, TM-Transmembrane domain). These vectors were co-transfected with 6myc-ARTS into Bcl-2 KO MEFs. IP of ARTS was performed in MEFs transfected with Bcl-2-BH deletions, WT Bcl-2 (positive control) and empty vector (negative control). A significant reduction in binding of ARTS to Bcl-2delBH3 was seen. See also supplemental Tables S1 and S2. C. BIFC assays were performed as described in Figure 3C. HeLa cells were co-transfected with vectors expressing ARTS, Bcl-2 and Bcl-2delBH3 fused to either VN or VC parts of the YFP Venus fragment (ARTS-VN, Bcl-2-VC. Bcl-2delBH3 –VC). A 47% decrease in the proximity between ARTS and Bcl-2delBH3 was seen compared to WT Bcl-2. Jun/bFos served as positive control (p.c.), and Jun/bFosdel ZIP as negative control (n.c.). Results are shown as mean + S.E. of three independent experiments. (*, p ≤ 0.05; **, p ≤ 0.01). See also supplemental Figure S3. D. In vivo ubiquitylation of Bcl-2 WT and Bcl-2delBH3. MEFs Bcl-2 KO cells were transfected with Flag-Bcl-2WT or Flag-Bcl-2delBH3. Cells were treated with MG132 and STS for one hour or left untreated. Proteins were immunoprecipitated with anti-ubiquitin. Poly-ubiquitylated forms of Bcl-2 were detected using an anti-Bcl-2 antibody. While WT Bcl-2 undergoes ubiquitylation following treatment with STS, no ubiquitylation of Bcl-2delBH3 was seen. See also supplemental Figure S4.