FIGURE 6.

Stable expression of mOpsin·Rluc and mOpsin·Venus in HEK-293 cells. A stable cell line, namely HEK-293 (mOpsin·Rluc and mOpsin·Venus), was generated by sequential genome integration of mOpsin·Rluc and mOpsin·Venus. A, expression of mOpsin·Rluc and mOpsin·Venus was assessed in 50 μg of total protein cell lysate by immunoblotting with a monoclonal antibody recognizing the N terminus of Rho (B6-30) and a polyclonal antibody against Rluc, respectively. The ratio of mOpsin·Rluc to mOpsin·Venus was calculated to be 1:2.4. Note that the sample was run on the same gel and transferred onto PVDF membranes developed individually with different antibodies. B, membrane localization of mOpsin·Venus was determined by detecting Venus fluorescence, and mOpsin·Rluc was detected by immunostaining with anti-Rluc antibody. Merging of the two images indicates co-localization of both receptors. C, specificity of the BRET signal. Left, light gray vertical bars: the BRET signal was recorded in HEK-293 cells transiently transfected with vectors expressing mOpsin·Rluc (donor) only or mOpsin·Rluc (donor) with Kras·Venus (acceptor) used as a negative control (here the BRET increase was due to co-localization of the BRET donor and acceptor on cell membranes), and mOpsin·Rluc (donor) and mOpsin·Venus (acceptor) (in this case, the BRET increase was due to Rho dimerization, an effect greater than that achieved by co-localization). Right, dark gray vertical bars: the BRET signal was recorded in the stable HEK-293 (mOpsin·Rluc and mOpsin·Venus) cell line before and after treatment with 1 mm DDM. D, the decrease of the BRET signal with increasing concentrations of DDM shown is due to the disruption of opsin dimers.