Extended Data Fig. 7: In-gel digestion proteomics and native mass spectra of G_t and PDE6 complexes isolated from dark-adapted rod outer segments under different conditions.

a, Separation of the component subunits of the Gt and PDE6 complex on a 2D page gel, pre-stained with protein standards. b, Unique peptides of Gα1 (GNAT1) and c, Gα2 (GNAT2) are identified by proteomics (see Methods). d, Gt with rho-containing membrane in the absence of light reveals the initial extent of endogenous GDP binding to Gt (blue and green for γ¹ and γ² isoforms respectively) and without GDP (pink and yellow). e, Gt preparation illuminated in the absence of membranes was found to be bound to GDP predominantly for both isoforms. No reaction is observed in the absence of the membrane. f, with rho in membranes following illumination, GDP bound forms have declined in favour of apo states (pink and yellow). g, with endogenous levels of GTP, the PDE6:PDE6•cGMP complex remains ~ 1:1 after activation of Gt though rho*. The endogenous quantity of GTP is insufficient to produce sufficient Gtα._GTP to interact with PDE6 and promote hydrolysis of cGMP. h, in the presence of an additional molar amount of GTP, G_t•GDP is replenished and further cGMP hydrolysis takes place. i, in the presence of an additional molar aliquot of GTPγs further cGMP hydrolysis has occurred but G_t•GDP is not replenished. The SDS-page electrophoresis (a) was performed twice; in-gel proteomics experiment (b, c) was performed once and the real-time native MS experiments (d–f) and bulk reactions (g–i) were repeated three times and twice respectively.