Figure 1.

Neurons that overexpress Par-4 show an exacerbated rate of cell death following the mechanical insult compared to wild-type neurons. A. Representative Western blotting analysis showing high level expression of Par-4 protein in cortical neurons from transgenic mice. Primary cultures of cortical neurons were established and western blot analyses were performed in 7-day old cultures using a monoclonal antibody raised against a recombinant protein corresponding to amino acids 1-334 representing full length PAR4 of rat origin (Santa Cruz Biotechnology, Inc.). Relatively high levels of Par-4 protein were detected in neurons from Par-4 transgenic mice (lane 2), while only minimal levels of endogenous Par-4 were observed in normal neurons from wild-type mice (lane 1). B. Genotyping of Par-4 transgenic mice using a PCR-based protocol. We have developed a quick PCR-based assay of the DNA from tail biopsies to amplify a 236-bp simian virus 40 fragment from pNSE-Par4 vector, which is detectable only in mice transgenic for Par-4 (lanes 1-3), but not in wild-type mice (lanes 4-6). C. Phase contrast images of cortical neurons from wild-type and Par-4 transgenic mice before and after injury. Overall neuron survival decreases at each time point. D. Graph shows percentage of surviving cells at each time point as indicated: uninjured (Un), immediately after injury (0), 4, 8, 12 and 24 hrs after injury. A higher percentage of cell death was observed in Par-4 transgenic cortical neurons compared to wild-type neurons. Values are the means and standard derivations of determinations from eight separate cultures (20-30 neurons analyzed per culture). ***p<0.001 compared with corresponding values in cells from wild-type mice. ANOVA with Scheffe's post-hoc tests.