Fig. 5.
Fig. 5. Functional analysis of region I, II, and III mutations in the type 2 CD10/NEP promoter. (Left) Mutated CD10/NEP(−263/+147)pXP2 constructs. Wild-type CD10/NEP (−263/+147)pXP2 construct (WT) and mutated region I (I-M), II (II-M), and III (III-M) constructs are shown. (Right) Luciferase assays of mutated CD10/NEP(−263/+147) constructs. The indicated WT and mutated CD10/NEP (−263/+147)pXP2 constructs were cotransfected with CMV-GH plasmid into FHTE56 and Nalm-6 cells. Resulting luciferase activities were measured and normalized for transfection efficiency by standardizing for GH production driven by the CMV-GH internal control. Promoter activity (RLU) represents the mean ± SE for two separate experiments performed in duplicate: FHTE56 v Nalm-6: WT, 1,293 ± 123 (100%) v 245 ± 12 (100%); I-M, 721 ± 69 (56%) v 257 ± 18 (105%); II-M, 774 ± 57 (60%) v 162 ± 8 (66%); III-M, 1,716 ± 160 (133%) v 357 ± 18 (146%). Differences between promoter activity driven by WT and mutated constructs were evaluated using a one-sided Student's t-test.

Functional analysis of region I, II, and III mutations in the type 2 CD10/NEP promoter. (Left) Mutated CD10/NEP(−263/+147)pXP2 constructs. Wild-type CD10/NEP (−263/+147)pXP2 construct (WT) and mutated region I (I-M), II (II-M), and III (III-M) constructs are shown. (Right) Luciferase assays of mutated CD10/NEP(−263/+147) constructs. The indicated WT and mutated CD10/NEP (−263/+147)pXP2 constructs were cotransfected with CMV-GH plasmid into FHTE56 and Nalm-6 cells. Resulting luciferase activities were measured and normalized for transfection efficiency by standardizing for GH production driven by the CMV-GH internal control. Promoter activity (RLU) represents the mean ± SE for two separate experiments performed in duplicate: FHTE56 v Nalm-6: WT, 1,293 ± 123 (100%) v 245 ± 12 (100%); I-M, 721 ± 69 (56%) v 257 ± 18 (105%); II-M, 774 ± 57 (60%) v 162 ± 8 (66%); III-M, 1,716 ± 160 (133%) v 357 ± 18 (146%). Differences between promoter activity driven by WT and mutated constructs were evaluated using a one-sided Student's t-test.

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