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. Author manuscript; available in PMC: 2009 Jul 27.
Published in final edited form as: Invest Ophthalmol Vis Sci. 2008 Nov 21;50(4):1769–1777. doi: 10.1167/iovs.08-2909

FIGURE 1.

FIGURE 1

Downregulation of ponsin expression in Rho GTPase–targeted mouse lenses. In addition to cDNA microarray data, the downregulation of ponsin expression in Rho GDIα transgenic mouse lenses was confirmed by semiquantitative (A) and quantitative (B) RT-PCR analyses and by immunoblot analysis (C). Equal amounts of total RNA derived from the P7 lenses (pooled) of Rho GDIα transgenic and littermate wild-type mice were reverse transcribed. Complementary DNA from the RT reaction was subjected to semiquantitative and quantitative PCR analyses with two different ponsin sequence-specific oligonucleotide primer sets. (A) The two different ponsin-specific oligonucleotide primer sets used for PCR analysis amplified more than one splice variant of ponsin, and with both sets of primers, the expression of one of the ponsin variants (top bands) was found to be markedly decreased in Rho GDIα transgenic lenses. Contrarily, one of the ponsin variants (bottom band with first primer sets) was found to be marginally increased in the Rho GDIα transgenic lenses compared with wild-type lenses. Expression of G3PDH was used as an internal control to normalize the cDNA content in the PCR reactions. (B) Quantitative PCR analysis of ponsin expression using two different oligonucleotide primer sets revealed an obvious decrease in Rho GDIα transgenic lenses compared with wild-type lenses. The q-PCR traces from duplicate reactions are depicted. (C) Immunoblot analysis of ponsin isoforms in lenses derived from Rho GDIα– and C3 exoenzyme (Rho GTPase inactivating bacterial toxin)– overexpressing transgenic mice. The water-insoluble fraction derived from pooled P7 lenses was subjected to immunoblot analysis with a polyclonal anti-ponsin antibody that reacts with different CAP/ponsin isoforms. In both the Rho GDIα transgenic and C3 exoenzyme– overexpressing transgenic lenses, protein levels of ponsin isoforms were found to be markedly decreased compared with wild-type lenses. Compared with Rho GDIα–transgenic lenses in which the >90-kDa (arrow) immunopositive band was decreased dramatically, several different ponsin immunopositive protein bands were found to be decreased in addition to the 90-kDa isoform in the C3 transgenic lenses. These results were based on duplicates of pooled lenses.