Construction of plasmid and adenovirus vectors. PCR to construct pENTR vectors was performed using PrimeSTAR DNA polymerase (Takara) and primers (Sigma–Aldrich). A series of Mlx MGCD 0103 mutants with an N-terminal Flag tag were amplified from mouse liver cDNA ( Fig. 1A). PCR fragments (2–245aa, 67–245aa, and 88–245aa) were cloned into the pENTR vector (Invitrogen), yielding pENTR-Mlx1-1, -Mlx2-1, or -Mlx4-1, respectively. Adenovirus vectors bearing Mlx4-1 were constructed by recombining pENTR-Mlx4-1 vectors into pAd/CMV/V5-DEST using LR Clonase II Master Mix (Invitrogen) according to the manufacturer’s protocol. The pcDNA6.2 vectors bearing Mlx1-1, Mlx2-1, or Mlx4-1 (pcDNA-Mlx1-1, pcDNA-Mlx2-1, or pcDNA-Mlx4-1, respectively) were constructed in the same manner as the adenovirus. pGL3-Lpk and Ad-daChREBP were the same vector and adenovirus used previously [5]. pGL4-TK-RLuc vector was purchased from Promega.
Fig. 1.
(A) Schematic representation of wild-type Mlx (Mlx1-1), dominant active Mlx (Mlx2-1), and dominant negative Mlx (Mlx4-1). (B) Effects of Mlx1-1, Mlx2-1, or Mlx4-1 on Lpk promoter activity. Isolated hepatocytes were transfected with 3.6 μg of pGL3-Lpk, 0.4 μg of pGL4-TK-RLuc, and 0.4 μg of pcDNA6.2 empty, Mlx1-1, Mlx2-1, or Mlx4-1 vector using Lipofectamine 2000. Transfected cells were cultured for 24 h and used for analysis of luciferase activity. Data are means ± S.D. (n = 6 per group). ?,??p
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