Fig. 2: Bcl-xL, but not its BH4 domain, binds to IP3R1 involving LBD and Fragment 3.
From: Bcl-xL acts as an inhibitor of IP3R channels, thereby antagonizing Ca2+-driven apoptosis
a Representative co-immunoprecipitation experiments using anti-FLAG performed in lysates from HeLa cells transiently overexpressing 3xFLAG-Bcl-2 or 3xFLAG-Bcl-xL. This experiment was performed three times using each time independently transfected and freshly prepared cell lysates. The samples were analyzed via western blot using antibodies against IP3R1 and FLAG. Total HeLa lysates were used as input (20 µg). PD: pull down; IB: immunoblot. b Linear representation of a mouse IP3R1 (mIP3R1) monomer. The three functional domains, including the ligand-binding domain (LBD), and the five tryptic fragments, including Fragment 3, are represented. Respective amino acids are indicated by numbers. TMDs: transmembrane domains. c Representative GST-pull down experiment for assessing the binding of 3xFLAG-Bcl-xL from COS-7 cell lysate to GST-fused IP3R1 fragments. The samples were analyzed via western blot. Total COS-7 lysate was used as input (0.1 µg). This experiment was performed four times utilizing each time independently transfected and freshly prepared cell lysates. PD: pull down; IB: immunoblot. The corresponding western blot for the GST-IP3R fragments was shown in Fig. S2a. d Binding curves showing the interaction of purified 6xHis-Bcl-xL with titrated GST-fused IP3R domains generated by MST. GST was used as a negative control. Concentration of the 6xHis-Bcl-xL was kept constant at 50 nM, whereas the GST-LBD, GST-Fragment 3, GST-Fragment 5b and parental GST proteins were titrated down from 15 µM to 5 nM. The unit of the left axis (ΔFnorm) is a ratio of normalized fluorescence. Data points represent mean ± SD from triplicate measurements. e Representative sensorgrams of SPR experiments showing the binding properties of GST-fused IP3R-LBD, applied at 1.1 µM, to biotin-BH4-Bcl-xL and biotin-BH4-Bcl-2 peptides. The biotin-BH4 peptides, immobilized on a streptavidin-coated sensor chip. Sensorgrams were obtained after background correction for binding to the scrambled peptides. Data are expressed in resonance units (R.U.) as a function of time. f Quantitative analysis of the binding properties of biotin-BH4-Bcl-2 and biotin-BH4-Bcl-xL peptides to GST-LBD measured by SPR. Values obtained from independent experiments were plotted as mean ± SEM (N = 4).
