(A) Ligand-induced Smad1–Smad5 and Smad2–Smad3 interactions. COS-7 cells were cotransfected with CBRN-Smad1, Smad5-McLuc1, ELucN-Smad2 and Smad3-McLuc1 and their bioluminescence intensities were obtained with a luminometer. (*P<0.05) (B) Analysis of bioluminescence with different combination of Smads. COS-7 cells were cotransfected with either CBRN-Smad1 and Smad3-McLuc1 or ELucN-Smad2 and Smad5-McLuc1. Representative data were shown.

The CHO-IR cells were transiently transfected with IRS1-McLuc1 and FLucN-p85β; luciferase activities were measured at each time point. Error bars represent s.d. calculated for three independent samples.

Structural models of each luciferase (upper part of each image) are based on the X-ray crystal structure of full-length Photinus pyralis (Firefly) luciferase. The N-terminal and C-terminal domains of respective luciferases are shown in different colors. Bioluminescence images of COS-7 cells transfected with plasmids expressing the protein fragments fused to FKBP and FRB are shown below the luciferase structures. The cells were cultured on the 24-well plate and incubated in the presence (+Rap) and absence (−Rap) of rapamycin.

(A) Characterization of the probes. a and b: COS-7 cells were transfected with the plasmids, Smad4-McLuc1 plus FLucN-Smad1 or Smad4-McLuc1 plus CBRN-Smad1, in the absence (Mock) and presence of either ALK3CA or ALK3DN receptor. Smad1(AXA) and Smad2(AXA) indicate double mutants of Smad1(S462A,S464A) and Smad2(S465A,S467A), respectively. The cells were incubated for 12–16 h and the luciferase activities were measured. c: COS-7 cells were transfected with the plasmids, ELucN-Smad2 plus Smad4-McLuc1 or ELucN-Smad2(AXA) plus Smad4-McLuc1 in the absence (Mock) and presence of either ALK5CA or ALK5DN receptor. d: COS-7 cells transfected with the plasmids FLucN-Smad1 and Smad4-McLuc1 were stimulated with BMP-2 (50 nM) for 2 h and luciferase activities were measured. Error bars represent s.d. calculated for three independent samples. e: Western blotting analysis of the expression of Smad1–Smad4 or Smad2–Smad4 protein in the presence of ALK3CA, ALK3DN, ALK5CA or ALK5DN. (*P<0.05, **P<0.01, ***P<0.001) (B)–(D) Real-time bioluminescence images of Smad1–Smad4 and Smad2–Smad4 interactions using CBRN-Smad1 and Smad4-McLuc1 (B), CBRN-Smad2 and Smad4-McLuc1 (C), and CBRN-Smad1, ELucN-Smad2 and Smad4-McLuc1 (D), in a Xenopus embryo. RNAs encoding the Smad probes were injected into the animal pole of a 2-cell stage Xenopus embryo. The embryo was incubated for 24 h at 13°C and thereafter, digital images of Venus (gray) and bioluminescence (pseudocolor) were acquired using a microscopic system equipped with an EM-CCD camera. Bar, 1 mm.

(A–C; upper) Absolute photon counts for COS-7 cells cotransfected with the plasmids of a pair of luciferase fragments fused to FKBP and FRB in the absence (white bars) and presence (black bars) of rapamycin. (A–C; lower) Normalized photon counts for COS-7 cells transiently cotransfected with plasmids expressing the luciferase fragments fused to FKBP and FRB, and Renilla luciferase. The Renilla luciferase was used to normalize the transfection efficiency. Results are expressed as the relative luminescence unit (RLU) ratio; values of which the luminescence intensity were normalized to the intensity of Renilla Luciferase for rapamycin-treated cells were divided by those for rapamycin-untreated cells (black bars). Differences of heights between white and black bars indicate rapamycin-induced luminescence. (D) Reversibility of the complementation between McLuc1 and N-terminal FLuc. The lysates extracted from COS-7 cells expressing FKBP and FRB fused to the luciferase fragments were treated with rapamycin for 1–10 min (50 nM, upper data). The cells were treated successively with different concentrations of FK506 (1 µM, 10 µM, 100 µM) for 0–25 min in the presence of 50 nM rapamycin (middle data). Photon counts were taken every 60 s. The luminescence values were normalized against the maximum luminescence values. Western blotting analysis of the cells including LucN and McLuc1 in the presence or absence of rapamycin and FK506 with cycloheximide (lower). Error bars represent s.d. calculated for three independent samples. (**P<0.01, ***P<0.001)

(A) Results of analyses of interactions between 14-3-3 and Bad mutants. The COS-7 cells were transiently transfected with Bad–McLuc1 and ELucN–14-3-3; luciferase activities were tested. Error bars represent s.d. calculated for three independent samples. (B) Results of Western blotting analysis of Bad phosphorylation. Expression levels of Bad and its mutants were determined using immunoblotting with the anti-V5 antibody (left). Mutations of Bad at S112A, S136A, and S155A were confirmed by immunoblotting with the respective antibodies (right). (C) An inhibitory effect of antimycin or HA14-1 on the bioluminescence was developed by the Bad-Bcl-2 interaction. Antimycin (10 µM) or HA14-1 (10 µM) was added to the cells after transfection and incubated for 20 h. The cells were harvested and the photon counts were analyzed. Error bars represent s.d. calculated for three independent samples. (D) Dual color imaging of mice with a single substrate. The images shown are superimposed on the optical CCD bioluminescence image without a filter (Open) or with a band-pass filter of BP(ELuc) (525±25 nm) or BP(SLRLuc) (630±37.5 nm). A nude mouse was imaged after implantation of COS-7 cells that had been transiently transfected with plasmids SLRLuc (site 1), Bad–McLuc1 and ELucN–14-3-3 (site 2), SLRLuc plus Bad–McLuc1 and ELucN–14-3-3 (site 3), and SLRLuc plus Bad(S112A, A136A, A155A) –McLuc1 and ELucN–14-3-3 (site 4). To obtain photon flux information from mice, the bioluminescence intensity was shown as pseudocolors. Photon counts with BP(ELuc) divided by those with BP(SLRLuc) are shown at the right side of the image.