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. 2011 Jan 30;4(2):124–133.

Figure 2.

Figure 2

JunB promotes GzB transcription in ALK+ ALCL. A. qRT-PCR analysis of GzB mRNA levels in Karpas 299 (left) and SUP-M2 (right) cells transfected with 100 nM of the indicated pooled siRNAs. Note: these experiments were performed on the mRNA extracted from cells transfected in Figure 1. B. Luciferase activity was measured in Karpas 299 cells transfected with a promoter-less luciferase construct (pGL2 basic) or a luciferase construct under control of the GzB proximal promoter (pGL2-GzB promoter) and 100 nM of the indicated pooled siRNAs. Luciferase activity is expressed relative to the activity present in cells co-transfected with the pGL2-GzB promoter and control siRNA (which was set at 100%). A western blot demonstrating the efficiency of JunB knock-down is shown to the right. C. Karpas 299 cells were transfected with the pGL2 basic, pGL2-GzB promoter, or AP-1 mutant pGL2-GzB promoter luciferase constructs and luciferase activity was measured 24 h post-transfection. Luciferase activity is expressed relative to the activity present in the pGL2-GzB promoter transfected cells (which was set at 100%). D. Luciferase activity was measured in Karpas 299 cells transfected with the pGL2-GzB promoter luciferase construct and Flag-tagged JunB (+) or empty vector (-) (top). Luciferase activity is expressed relative to the activity present in the pGL2-GzB promoter and empty vector–transfected cells (which was set at 100%). Anti-Flag and anti-JunB western blots demonstrate JunB over-expression (bottom). Molecular mass markers are indicated to the left of blots. E. EMSAs were performed with Karpas 299 nuclear extracts using a biotinylated probe based on the sequence of the AP-1 site in the GzB proximal promoter (GzB AP-1 probe). For competitor experiments, a 200-fold molar excess of unlabeled GzB AP-1 probe (wt competitor) or an unlabeled GzB AP-1 probe with a mutation in the AP-1 binding site (AP-1 mutant competitor) were included in the reaction. For super-shift experiments, 1 µg of the indicated antibody (Ab) was included in the reaction. In all experiments, the quantification represents the mean and standard deviation of three independent experiments. p values were determined using paired, one-tailed t-tests.