Figure 2.

Genotype and ADP-ribosylation patterns of wild-type and α_o−/− mice. (A) Southern blot analysis of XhoI-digested DNA from tail biopsies of −/− mice and +/− and +/+ littermates; the probe was the 3′ external probe shown in Fig. 1. (B) Selected tissues from wild-type mice and mice predicted by Southern blot analysis to be G_o-deficient. Homogenizations were all in 27% sucrose/1 mM EDTA/10 mM Tris⋅HCl, pH 7.5. Homogenates were ADP-ribosylated without further processing in the presence of guanosine 5′-[β-thio]diphosphate, adenosine 5′-[β,γ-imido]triphosphate, 0.1% SDS, and 2 mM DTT for 30 min at 32°C in a final volume of 15 μl as described (36). NAD was then added to give 4 mM, mixed with 30 μl of 2× Laemmli’s sample buffer, and separated by urea-gradient SDS/PAGE in 9% gels (36). The gels were stained and destained over night and autoradiographed. Except for brain, the predominant bands are α_i2. Note that in the pituitary the intensities of α_o1 and α_o2 (above and below α_i2) are about equivalent; in brain, α_o1 is the predominant band with α_i1/3 and α_i2 being similar in intensity. In heart ventricle homogenates, only the α_i2 band is evident [absent in α_i2-deficient hearts (37)].