Acknowledgments

The work reviewed in this paper was supported by the National Institutes of Health, USA, National Institute of Neurological and Communicative Disorders and Stroke (Steve Heinemann and Jim Patrick), and the Muscular Dystrophy Association of America (Steve Heinemann and Jim Patrick).

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Figure 4 continued with proteinase K(10ug/ml, 37°C, 30 minutes), acetylated, and dehydrated. Hybridization with 35S-radiolabeled RNA probe (5-10 x 10 cpm/ml) was performed at 55°C for 12-18 hrs in a solution containing 50 percent formamide, 0.3M NaCl, 10mM Tris(pH 8.0), 1mM EDTA, 0.05 percent tRNA, 10mM DTT, 1x Denhardt's solution, and 10 percent dextran sulfate. Because of the high sequence similarities in the protein coding regions of the cDNAs, 3' untranslated sequences were used to make probes. The EcoRI/3' end, Ball/3' end, and BglI/3′ end fragments derived from C183, PCA48, and a4-2 cDNA clones, respectively, were subcloned into the plasmid PSP65 and used to synthesize antisense RNA probes in vitro. After hybridization, sections were treated with RNaseA (20μg/ml, 37°C, 30 minutes) and washed in 0.1 x SSC at 55°C. Dehydrated slides were exposed to x-ray films for 3-16 days at 4°C. An RNA probe coding the sense strand of C183 clone was used as a control.

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