Fig. 8.
Fig. 8. FOG acts as a negative (inhibitory) cofactor for GATA-1 transactivation of the MBP promoter. (A) Northern blot analysis of mRNA for FOG in uninduced HT93A cells (lane 1) and HT93A cells induced with RA for 1, 3, 5, and 7 days (lanes 2 through 5, respectively). Control hybridization with β-actin cDNA is shown in the lower panel. (B) FOG inhibits transactivation of the MBP promoter by GATA-1. Jurkat cells were transiently transfected with 5 μg of pXP2-MBP, 2 μg of pEF-GATA-1, increasing amounts of pMT2-FOG as shown, and 1 μg of β-galactosidase expression vector (pCMV-βGal). Data are shown as the mean of 3 independent experiments (±SEM). (C) Jurkat cells were transfected by the electroporation method with 2 μg of the pXP2-MBP containing wild-type GATA- and C/EBP-binding sites along with the following expression vectors: 4 μg of pEF-BOS, 2 μg of pEF-GATA-1 plus 8 μg of pMT2, 2 μg of pEF-GATA-1 plus 7 μg of pMT2 plus 1 μg of pMT2-FOG, 2 μg of pEF-GATA-1 plus 6 μg of pMT2 plus 2 μg of pMT2-FOG, 2 μg of pEF-GATA-1 plus 4 μg of pMT2 plus 4 μg of pMT2-FOG, and 2 μg of pEF-GATA-1 plus 8 μg of pMT2-FOG. pBluescriptIIKS(−) plasmids were added to maintain total DNA constant at 22 μg. Luciferase activity was measured 24 hours after transfection and normalized for transfection efficiency based on the activity of cotransfected β-galactosidase expression vector (pCMV-βGal). (D) Jurkat cells were transfected with 5 μg of the pXP2-MBP containing a mutated GATA-binding site (pXP2-MBP mutGATA) along with the following expression vectors: 2 μg of pEF-BOS, 2 μg of pEF-C/EBPβ, 2 μg of pEF-C/EBPβ plus 7 μg of pMT2 plus 1 μg of pMT2-FOG, 2 μg of pEF-C/EBPβ plus 6 μg of pMT2 plus 2 μg of pMT2-FOG, 2 μg of pEF-C/EBPβ plus 4 μg of pMT2 plus 4 μg of pMT2-FOG, and 2 μg of pEF-C/EBPβ plus 8 μg of pMT2-FOG. pBluescriptIIKS(−) plasmids were added to maintain total DNA constant at 22 μg. Luciferase activity was measured 24 hours after transfection and normalized for transfection efficiency based on the activity of a cotransfected β-galactosidase expression vector (pCMV-βGal). Data are shown as the mean of 3 independent experiments (±SEM).

FOG acts as a negative (inhibitory) cofactor for GATA-1 transactivation of the MBP promoter. (A) Northern blot analysis of mRNA for FOG in uninduced HT93A cells (lane 1) and HT93A cells induced with RA for 1, 3, 5, and 7 days (lanes 2 through 5, respectively). Control hybridization with β-actin cDNA is shown in the lower panel. (B) FOG inhibits transactivation of the MBP promoter by GATA-1. Jurkat cells were transiently transfected with 5 μg of pXP2-MBP, 2 μg of pEF-GATA-1, increasing amounts of pMT2-FOG as shown, and 1 μg of β-galactosidase expression vector (pCMV-βGal). Data are shown as the mean of 3 independent experiments (±SEM). (C) Jurkat cells were transfected by the electroporation method with 2 μg of the pXP2-MBP containing wild-type GATA- and C/EBP-binding sites along with the following expression vectors: 4 μg of pEF-BOS, 2 μg of pEF-GATA-1 plus 8 μg of pMT2, 2 μg of pEF-GATA-1 plus 7 μg of pMT2 plus 1 μg of pMT2-FOG, 2 μg of pEF-GATA-1 plus 6 μg of pMT2 plus 2 μg of pMT2-FOG, 2 μg of pEF-GATA-1 plus 4 μg of pMT2 plus 4 μg of pMT2-FOG, and 2 μg of pEF-GATA-1 plus 8 μg of pMT2-FOG. pBluescriptIIKS(−) plasmids were added to maintain total DNA constant at 22 μg. Luciferase activity was measured 24 hours after transfection and normalized for transfection efficiency based on the activity of cotransfected β-galactosidase expression vector (pCMV-βGal). (D) Jurkat cells were transfected with 5 μg of the pXP2-MBP containing a mutated GATA-binding site (pXP2-MBP mutGATA) along with the following expression vectors: 2 μg of pEF-BOS, 2 μg of pEF-C/EBPβ, 2 μg of pEF-C/EBPβ plus 7 μg of pMT2 plus 1 μg of pMT2-FOG, 2 μg of pEF-C/EBPβ plus 6 μg of pMT2 plus 2 μg of pMT2-FOG, 2 μg of pEF-C/EBPβ plus 4 μg of pMT2 plus 4 μg of pMT2-FOG, and 2 μg of pEF-C/EBPβ plus 8 μg of pMT2-FOG. pBluescriptIIKS(−) plasmids were added to maintain total DNA constant at 22 μg. Luciferase activity was measured 24 hours after transfection and normalized for transfection efficiency based on the activity of a cotransfected β-galactosidase expression vector (pCMV-βGal). Data are shown as the mean of 3 independent experiments (±SEM).

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal