Three genes, MafK, SytI, and Syn 1, in the fifth group of clustering drew our interest because these genes are known or presumed to be associated with neuronal function. MafK is one of BTB06584? the small Maf family proteins. It is expressed in neural tissues of both late embryonal stage and postnatal period, in the embryonic mesoderm, Inhibitors,Modulators,Libraries and in mesenchymal and hematopoietic cells. MafK certainly plays a role in differentiation of these specific types of cells. Inhibitors,Modulators,Libraries it participates in NGF promoted neuritogenesis of PC12 cells and imma ture telencephalon neurons, and is required for eryth roid differentiation. SytI is abundant in the synaptic vesicles of neurons, where it acts as a calcium sensor and plays a critical role in neurotransmitter release.
Its expression was shown to be induced in neural crest cultures by BMP4, a factor Inhibitors,Modulators,Libraries that evokes noradrenergic differentiation. Syn 1 is involved mainly in the regulation of plasma membrane dynamics in neuronal as well as non neuronal cells. Ectopic expression of Syn 1 in neu rons was shown to increase the number of neuritic varicosi ties. We performed quantitative RT PCR to quantitate the dif ferences between transcript levels in three cell populations PC12, PC12, and PC12. The transcript level of each gene in PC12 and PC12 cells was presented as a fold change from that of the PC12 cells. As shown in Fig. 2A, all of the three genes exhibited increased transcript levels in Inhibitors,Modulators,Libraries both PC12 and PC12 cells compared to PC12 cells. The transcript levels of these genes seem inversely correlated with the Akt activity levels, being highest in PC12 and lowest in PC12.
The SytI transcript was remarkably elevated, with an 18 fold increase in PC12 cells, compared to PC12 cells. The fold differences of other gene Inhibitors,Modulators,Libraries tran scripts were 2. 7 4. 3. To check whether detected changes in the transcript levels are followed by changes in protein lev els, western blot analysis was performed. In agreement with the transcript levels, MafK, SytI, and Syn 1 protein levels were also higher in PC cells than in PC cells. As stated above, our experiments were based on two Akt manipulated PC12 sublines that turned out to have quite different neurite outgrowth phenotypes from each other. cells overexpressing Akt do not differentiate well but the other cells that express the dominant negative form of Akt make neurites quite well. However, the specificity of domi nant negative Akt alleles has been contentious, and whether the elevated levels of gene transcripts in PC12 cells are due to increased mRNA stability in these Akt downregulated cells. Gene transcript sellekchem decay assays employing the transcriptional inhibitor actinomycin D revealed that the mRNA stability of MafK, SytI, and Syn 1 does not differ between PC12 and PC12 cells.