Activation of heterotrimeric G proteins by Smoothened -- Riobo et al. 103 (33): 12607 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Riobo et al. 10.1073/pnas.0600880103.

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Supporting Figure 5
Supporting Figure 6
Supporting Figure 7

Supporting Figure 5

Fig. 5. Cyclopamine does not interfere coupling of the 5-HT_1A receptor to G_i2. Membranes from Sf9 cells infected with baculoviruses encoding 5-HT_1A receptor, Ga_i2, Gb₁, and Gg₂ were preincubated for 10 min with 5 mM cyclopamine or vehicle and then incubated with 1 nM [³⁵S]GTPgS with or without 1 mM 8OH-DPAT for an additional 10 min. Ga_i2 was immunoprecipitated by using the Ga_i-directed antiserum, and radioactivity incorporated as bound [³⁵S]GTPgS was quantified by liquid scintillation spectrometry. The experiment is representative of two independent assays performed in triplicate.

Supporting Figure 6
Fig. 6.
Purmorphamine activates G_i2 through Smo. Membranes from Sf9 cells infected with baculoviruses encoding Smo, Ga_i2, Gb₁, and Gg₂ were preincubated with increasing concentrations of purmorphamine for 10 min and then incubated with 1 nM [³⁵S]GTPgS for an additional 10 min. Ga_i2 was immunoprecipitated by using the Ga_i-specific antiserum, and radioactivity incorporated as bound [³⁵S]GTPgS was quantified by liquid scintillation spectrometry. The broken line indicates the [³⁵S]GTPgS binding to Gi2 alone, i.e., in the presence of 5 mM cyclopamine.

Supporting Figure 7
Fig. 7.
Oncogenic SmoM2 activates G_i2. Membranes from Sf9 cells infected with baculoviruses encoding Gai₂, Gb₁, and Gg₂ alone or with a baculovirus encoding, or SmoM2, were preincubated for 10 min, and then incubated with 1 nM [³⁵S]GTPgS for an additional 10 min. Ga_i2 was immunoprecipitated by using the Ga_i-specific antiserum, and radioactivity incorporated as bound [³⁵S]GTPgS was quantified by liquid scintillation spectrometry. The experiment is representative of three independent assays.