FIG. 2.

Absence of constitutive activity of mORF74. (A) Activation of PLC. COS-7 cells were transfected with increasing amounts of cDNA encoding mORF74, 2 μg of cDNA encoding hORF74, or 5 μg of empty vector (mock). Forty-eight hours after transfection, InsP accumulation was determined. (Insert) Activation of PLC in the presence of PTX. COS-7 cells were transfected with 2 μg of cDNA encoding vGPCR or empty vector and incubated in the presence or absence of PTX. Forty-eight hours after transfection, InsP accumulation was determined. (B) Activation of NF-κB. COS-7 cells were transfected with increasing amounts of cDNA encoding mORF74, 2 μg of cDNA encoding hORF74, or 5 μg of empty vector (mock) and 5 μg of pNFκB-Luc. Forty-eight hours after transfection, NF-κB-driven luciferase expression was determined. (Insert) Activation of NF-κB in the presence of PTX. COS-7 cells were transfected with 2 μg of cDNA encoding vGPCR or empty vector and 5 μg of pNFκB-Luc and incubated in the presence or absence of PTX. Forty-eight hours after transfection, NF-κB-driven luciferase expression was determined. (C) Inhibition of cAMP. COS-7 cells were transfected with increasing amounts of cDNA encoding mORF74, 2 μg of cDNA encodinghORF74, or 5 μg of empty vector (mock) and 5 μg of pTLNC-21CRE and incubated in the presence or absence of PTX. Eighteen hours after transfection, forskolin (Fors) (10⁻⁵ M) was added. Twenty-four hours after transfection, CRE-driven luciferase expression was determined. (Insert) CRE modulation in the absence of forskolin. In all panels, a representative experiment performed in triplicate is shown. Each experiment was repeated at least two times.