Homogenates were cleared by centrifugation at 4 C in a microcentrifuge. Proteins were denatured by boiling in SDS PAGE sample buffer containing b mercaptoethanol and resolved by SDS PAGE. Proteins the following site were then electro phoretically transferred to PVDF membranes and probed with appropriate antibody. In these studies, immunoreactive bands were visualized by chemilumine sence using ECL plus western blotting detection system and captured on photographic film with subsequent digitization of images using a scanner. Inten sity of bands on digitized images was quantified using Imagequant TL. Choice of a protein for normalization was challenging for these studies because long term denervation resulted in profound alterations in levels of many cellular proteins, which was readily appreciated on Coomassie Blue stained SDS page gels of skeletal muscle lysates.

Commonly used housekeeping genes such as b tubulin, a actin and GAPDH proved to be unreliable for animals with prolonged denervation. Therefore, we have used the intensity of a neighboring non specific band for nor malization. Antibodies that were used in these studies include, RCAN2, FOXO1, REDD2, Apo D, and anti b tubu lin. Data are shown as mean SEM, and differences among means were determined by ANOVA as above. Statistics For real time PCR and western blotting data, differ ences among means were determined using one way ANOVA with a Newman Keuls multiple comparison test post hoc to test for significance of differences between pairs of means. Linear regression analysis was used to test correlations.

Calculations were performed using Graphad Prism 4. 0c. Background Flavonoid 3,5 hydroxylases and flavonoid 3 hydroxylases are versatile enzymes that accept several phenylpropanoid substrates. Of particular interest for anthocyanin pigmentation is the 3,5 or 3 hydroxylation of naringenin and dihydrokaempferol. F35Hs and F3Hs compete for substrate recruitment and deliver their 35 or 3 OH products into the paral lel synthesis of delphinidin and cyanidin, the precur sors of blue and red anthocyanins in grape berries, respectively. Variation in anthocyanin profile within and between grape varieties is associated with differences in the ratio of F35H to F3H expression. Anthocyanin biosynthesis takes place over 8 10 weeks, from shortly after berry softening until harvest.

F3Hs are expressed at com parable levels in both anthocyanin pigmented and green skinned varieties, before and after the onset of ripening. However, regulation of F35Hs is largely genotype specific and responsive to environmental cues. The breadth of diversity in fruit colour among dif ferent grapevine accessions suggests a fine regulation of Drug_discovery F35H expression. Dark blue cultivars transcribe F35Hs at higher levels than light red cultivars, which neverthe less maintain traces of 35 OH anthocyanins and barely detectable F35H transcripts. In green skinned cultivars, F35H transcripts are completely absent.