Figure 10

Effect of KLF15 over-expression on P1 minimal promoter activity. (A) Detection of KLF15 transcription factor in transiently transfected Rat2 cells. 48 hours after transfection of Rat2 cells with 25 to 500 ng/mL of pcDNA-hKLF15 or empty (mock) expression vector, nuclear (N) and cytoplasm (Cyt) proteins were extracted and 20 μg of total protein of each fraction were separated by SDS-PAGE. After transfer on nitrocellulose membrane, KLF15 was detected by chemiluminescence using anti-KLF15 antibody and horseradish peroxidase conjugated anti-goat IgG. Actin expression was used as a loading control. (B) Effect of KLF15 overexpression on fut8 P1 minimal promoter activity. Rat2 cells were co-transfected with 1 μg/mL of pGL3(-892/-451) vector containing the minimal promoter P1 sequence and 0 to 500 ng/mL of pcDNA-hKLF15 expression vector. 48 hours after transfection, firefly luciferase activity was measured and normalized by Renilla luciferase activity, as internal control. Histograms show a mean change in relative luciferase expression compared with control transfections, using the same concentration of empty vector (mock). ** P < 0.0005 (Student test). (C) EMSA analysis of nuclear proteins binding to (-892/-451) P1 promoter sequence. Lane 1, biotin 5'end-labeled (-892/-451) P1 promoter DNA probe; lane 2, labeled P1 promoter incubated with Rat2 cells nuclear proteins; lane 3, labeled P1 promoter incubated with Rat2 cells nuclear proteins in the presence of 200-fold molar excess of unlabelled sequence; lane 4, P1 promoter sequence incubated with nuclear proteins after pre-incubation with anti-KLF15 mAb. Back arrowhead indicates the electrophoretic mobility of biotin 5'end-labeled (-892/-451) P1 promoter DNA probe and open arrowhead the shift band obtained after incubation with Rat2 cells nuclear proteins.