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Fig. 5. A second siRNA targeting another region of the Cdc42 mRNA also upregulates MMP-1. Representative western-blot analysis of whole-cell lysates and of serum-free medium conditioned by HSFs 72 hours after transfection with 20 nM irrelevant siRNA (siScr), the first siRNA targeting Cdc42 (1st siCdc42) or the second siRNA targeting Cdc42 (2nd siCdc42), using specific antibodies to Cdc42, ERK1/2 and MMP-1. RT-PCR analysis of MMP-1 mRNA level and 28S rRNA was performed with total RNA extracted from HSFs cultured in DMEM containing 10% FCS 72 hours after transfection with 20 nM irrelevant siRNA (siScr), the first siRNA targeting Cdc42 (1st siCdc42) or the second siRNA targeting Cdc42 (2nd siCdc42). Sample-to-sample variations in RT-PCR efficiency are controlled using a known copy number of synthetic RNA co-transcribed and co-amplified with the same primers to generate a product of larger size (*).

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