Figure 2

REST/NRSF inhibits ICP22 and ICP4 promoter activity in transient cotransfection. A. Cotransfection of pICP22 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP22 promoter. 1. pICP22 and control plasmid. 2. pICP22 and pFLAG-REST (1:1). 3. pICP22 and pFLAG-REST (1:2). 4. pICP22 and pCMVp73 (1:1). 5. pICP22 and pCMVp73 (1:2). The asterisk P values represent Student's t tests in pairwise comparisons to the Lane 1 pICP22 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. B. Cotransfection of pICP4 with different amount of expression plasmids was performed followed by SEAP assay to analyze the regulatory effect of REST/NRSF on ICP4 promoter. 1. pICP4 and control plasmid. 2. pICP4 and pFLAG-REST (1:1). 3. pICP4 and pFLAG-REST (1:2). 4. pICP4 and pCMVp73 (1:1). 5. pICP4 and pCMVp73 (1:2). The P values represent Student's t tests in pairwise comparisons to the Lane 1 pICP4 + Control plasmid. The error bars represent standard deviations. The data were calculated and graphed using Microsoft Excel. C. Plasmid pSG28 and pH4-2 were cotransfected with pFLAG-REST or pCMVp73 followed by RNA isolation and RT-PCR. 1. pSG28 and control plasmid (1:1). 2. pSG28 and pFLAG-REST (1:1). 3. pSG28 and pCMVp73 (1:1). 4. pH4-2 and control plasmid (1:1). 5. pH4-2 and pFLAG-REST (1:1). 6. pH4-2 and pCMVp73 (1:1). D. The RT-PCR results were quantified by Kodak Gel-Logic 100 system to measure the sum intensity of each band representing the regulatory effect of REST/NRSF on ICP4 transcription.