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. 2012 May 25;7(5):e37702. doi: 10.1371/journal.pone.0037702

Figure 2. The truncated sequence of the Gas7 mutant is associated with control of Gas7 protein stability.

Figure 2

(A) Total RNA from the cerebrum of Gas7-deficient mice was subjected to RT-PCR followed by direct cloning of gas7 cDNA. Sequence analysis showed that a total length of 54 nucleotides (comprising exon 6 b) was deleted from the Gas7 wild-type allele. (B) The endogenous Gas7 mutant protein degraded faster than wild-type Gas7 in primary culture of cortical neurons. A Primary culture of cortical neurons was isolated at E16.5 from Gas7 wild-type or deficient mice. At DIV7, protein stability was analyzed with or without 100 µg/ml cycloheximide at the indicated time points. (C) The ectopically expressed Gas7 mutant protein degraded faster than wild-type Gas7 in 293 T cells. Overexpression of Gas7 wild-type and mutant protein in transiently transfected 293 T cells was analyzed by Western blot. 293 T cells were transfected with 0.5 µg Gas7 wild-type (WT) or 2 µg mutant (MT) plasmids, followed by culturing in the presence of 100 µg/ml cycloheximide, and harvesting at 0, 9, 18, 27, and 36 h after treatment. (D) The degradation rate of the Gas7 mutant protein is about 50% while the wild-type is about 15% at 27 hours. The results reflect the mean of triplicate experiments.