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. 2013 Nov 5;8(11):e78374. doi: 10.1371/journal.pone.0078374

Figure 2. Stimulation of promoter activity of the CCK gene by DJ-1.

Figure 2

A. Various deletion constructs of the CCK promoter linked to the luciferase gene were constructed and transfected into D2 cells together with pEF or pEF-DJ-1-HA. Forty-four hrs after transfection, cell extracts were prepared and their luciferase activity was measured as described in Materials and methods. n = 6. B. pGL3-CCK-1615 (WT) and reporter constructs of pGL3-CCK-1615 with deletion of recognition sites of either RREB1 (del- RREB1) or Sp1 (del-Sp1) and of both RREB1 and Sp1 (del-Sp1/RREB1) were transfected into NIH3T3 and D2 cells. Forty-four hrs after transfection, cell extracts were prepared and their luciferase activity was measured as described in Materials and methods. n = 4. C. NIH3T3 cells were transfected with RREB1 siRNA or with control siRNA. a) At 48 hrs after transfection, proteins were extracted from cells and analyzed by Western blotting with anti-RREB1 and anti-actin antibodies. b) At 48 hrs after transfection, total RNAs were extracted from cells, and the expression levels of CCK and actin mRNA were examined by real-time PCR, and relative expression of CCK versus actin is shown. c) At 24 hrs after siRNA transfection, pGL3-CCK-1615 (WT) was transfected into siRNA-transfected cells and their luciferase activities were measured at 48 hrs after transfection of pGL3-CCK-1615 (WT). n = 3. D. D2 cells in a 6-well dish were transfected with 0.75 µg of pSVP-Luc (Empty), pSVP-4xRREB1-wt (4xRREB1-wt) and pSVP-4xRREB1-mut (4xRREB1-mut) together with 0.25, 0.75 and 1.0 µg of pEF-DJ-1-HA. Forty-four hrs after transfection, cell extracts were prepared and their luciferase activity was measured. The expression level of DJ-1-HA in cell extracts was analyzed by Western blotting with an anti-HA antibody. n = 3.