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. 2011 May 6;18(11):1711–1725. doi: 10.1038/cdd.2011.47

Figure 7.

Figure 7

Binding of IPAS to pro-survival Bcl-2 family members through its C-terminal region. (a) Binding of IPAS to Bcl-xL but not to Bcl-2 and Bax. HEK293T cells were transfected with indicated combinations of expression plasmids and analyzed by immunoprecipitation with antibody against FLAG followed by immunoblotting with antibody against Myc. (b and c) Binding of IPAS to endogenous Bcl-xL but not to Bcl-2 and Bax. HEK293T cells were transfected with pBOS–3Myc–IPAS, incubated in the presence of Z-VAD-FMK and cell lysates were analyzed by immunoprecipitation with antibodies against rabbit IgG (Con.), Bcl-2, Bcl-xL and Bax followed by immunoblotting with antibody against Myc (b), or analyzed by immunoprecipitation with an antibody against Myc followed by immunoblotting with antibodies against Bcl-2, Bcl-xL and Bax (c). (d) Binding of IPAS to pro-survival Bcl-2 family members. Assays were carried out as in (a). (e) Binding of IPAS to Bcl-xL through its C-terminal region. Assays were carried out as in (a). (f) Subcellular localization of IPAS and Bcl-xL in HEK293T cells. HEK293T cells were transfected with pEGFP–IPAS constructs together with pBOS–3Myc–Bcl-xL plasmid, stained with antibody against Myc and observed using a confocal fluorescence microscope. (g) FLIM-FRET analysis of the interaction between IPAS-Cerulean (IC) and Bcl-xL-Citrine (BY) (left) and between IPAS C-Cerulean (ICC) and Bcl-xL-Citrine (right) in living CHO-K1 cells. CHO-K1 cells were transfected with indicated combinations of expression plasmids. The fluorescence decay curves of Cerulean (blue) represent an average of fluorescence decay data obtained from the cytoplasmic area of the observed cells. The decay curve of separately expressed IPAS-Cerulean or IPAS C-Cerulean (shown in black) was also shown. The shapes of the recorded instrumental response function (IRF) are shown in red. Data shown are representative of three independent experiments (top). FLIM images in the presence (IC+BY) or (ICC+BY) or absence (IC) or (ICC) of Bcl-xL-Citrine. Lifetime maps were made from TSCSPC data by fitting data to a single exponential decay. In the FLIM map, color corresponds to the fluorescence lifetime indicated by a false color scale (bottom). (h) Fluorescence decay data for IPAS-Cerulean or IPAS C-Cerulean in the presence or absence of Bcl-xL-Citrine in living CHO-K1 cells. a1 and a2 are the exponential coefficients for the τ1 and τ2 decay times, respectively. n: number of cells examined. The differences between the two τ1 values and the two τ2 values in the both case of IPAS WT and IPAS C were significant (P<0.001)