Figure 7.
HSPB6 competes with BCL2 binding to BECN1. (A and B), Reciprocal coimmunoprecipitations with the HSPB6 antibody and the BECN1 antibody revealed an interaction between HSPB6 and BECN1 in both NTG and HSPB6 WT TG hearts; (C to E), Recombinant human BECN1 protein was subject to a competitive ELISA containing increasing amounts of HSPB6 and BCL2, which revealed a competitive binding of HSPB6 and BCL2 with BECN1. BSA was used as a control. (C), A diagram for competitive ELISA to detect protein-protein interaction. High-binding 96-well ELISA plates were coated overnight with recombinant human BECN1 in carbonate buffer. Purified recombinant human BCL2 (D) or HSPB6 (E), as well as increasing amounts of HSPB6 (D) or BCL2 (E), were added and incubated for 2 h followed by 3 washes in PBST. Mouse monoclonal anti-BCL2 (D) or anti-HSPB6 (E) was used as a detection antibody and incubated for 2 h. Finally, tetramethylbenzidine substrate reagent was added, and absorbance was determined by a microplate reader at 450 nm. Three independent experiments were performed, values represent means ± SEM; *: P < 0.05, vs BSA; (F and G), Immunoprecipitation studies using BCL2 antibody as the bait and probed with BECN1 antibody in heart homogenates from HSPB6S10F TG mice and HSPB6 WT TG mice respectively, with NTG mice as controls. (F), Increased interaction of BECN1 with BCL2 in HSPB6S10F hearts; (G), Abolished interaction of BECN1 with BCL2 in HSPB6 WT hearts; 4 independent experiments were performed using 4 hearts for each group.