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. 2009 Jun 12;4(6):e5868. doi: 10.1371/journal.pone.0005868

Figure 6. Analysis of Bad phosphorylation based on luminescence intensities of the McLuc1 and N-terminal ELuc complementation.

Figure 6

(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.