Figure 7. NuBCPs disrupt Bcl-2’s intra-molecular interaction and binding with tBid.

(A) Intra-molecular interaction in Bcl-2. GFP-BH4 or Bcl-2/ΔBH4 was transfected alone or together into HEK293T cells. Their interaction was analyzed by Co-IP.

(B,C) Nur77 and NuBCP-9s disrupt Bcl-2 intra-molecular interaction. The indicated expression vectors were transfected into HEK293T cells. The effect of Nur77/ΔDBD and Nur77/DC3 (B) or NuBCP-9s (C) on interaction between BH4 domain and Bcl-2 was analyzed by Co-IP.

In A–C, input represent 5% of cell lysates used in the Co-IP assays, and representative data from one out of four independent experiments are shown.

(D) Removal of BH4 domain exposes BH3 domain. HEK293T cells transfected with either Bcl-2 or Bcl-2/ΔBH4 were stained with either anti-Bcl-2/BH3 or polyclonal anti-Bcl- 2 antibody. Scale, 10 µM.

(E) NuBCP-9 inhibits tBid/Bcl-2 interaction in liposomes. The membrane permeabilization induced by tBid/Bcl-2 interaction was monitored by the release of 0.5-kDa CB dyes from the liposomes after 3 hr incubation at 37 °C. The release resulted in the quenching of CB fluorescence by the anti-CB antibody located outside of the liposomes. The extent of the release was determined by the value of ΔF_Protein/ΔF_Triton as described in Supplemental Data. The effect of NuBCP-9 or NuBCP-9/AA peptide on the release was determined by including the corresponding peptide at indicated concentrations to the incubation. Data shown are means of 2 to 4 independent experiments with SD indicated by error bars.

(F) NuBCP-9-induced Bcl-2 conversion does not activate Bax pore activity in liposomes. The extent of the 0.5-kDa CB dye release by Bax and/or Bcl-2 in the absence or presence of NuBCP-9 peptide was monitored as above. Data shown are means of 2 to 5 independent experiments with SD (error bars).