Fig 1: Identification of active Fkh-binding sites in M. sexta moricin and lysozyme promoters. (A, B) Schematic diagrams of the truncated Mor-242 (A) and Lyz-345 (B) promoters. Fkh-1, 2, 3, 4 indicate the predicted Fkh-binding sites 1, 2, 3, 4; ?B and GATA-1 indicate the NF-?B and GATA-1 binding sites in the moricin promoter. Mut-1, -2, -3, -4 indicate the mutation of Fkh-binding sites 1, 2, 3, 4; Mut-1 & 2, Mut-1 & 3, Mut-2 & 3, and Mut-1, 2 & 3 indicate the mutations of Fkh-binding sites 1 and 2, 1 and 3, 2 and 3, as well as 1, 2 and 3, respectively. (C, D) Activation of Mor-242 and Lyz-345 promoters by recombinant DmFkh. The relative luciferase activities of Mor-242 and its Fkh-binding site mutant promoters (C), Lyz-345 and its Fkh-binding site mutant promoters (D) activated by recombinant DmFkh in S2 cells were determined by Dual-Luciferase® Reporter Assay System as described in the Materials and Methods. Bars represent the mean of three independent measurements ± SEM. For the relative luciferase activity among different promoters activated by DmFkh (comparing the solid bars), identical letters are not significant difference (p > 0.05) while different letters indicate significant difference (p < 0.05). For the activity of the same promoter after overexpression of DmFkh (comparing the solid and open bars for each promoter), the significance of difference was also determined by an unpaired t-test (*p < 0.05; **p < 0.01).
Fig 2: Moricin promoter is activated mainly by MsRel2-RHD.The relative luciferase activities of different truncated moricin promoters (A) or different NF-?B-deleted or mutated moricin promoters (B) activated by recombinant MsDorsal-RHD (Dl-RHD) or MsRel2-RHD (Rel2-RHD) in Sf9 cells, or different constructs of moricin promoters (see panel E) activated by recombinant MsDorsal-RHD, MsRel2-RHD, or the two RHDs together (Dl + Rel2) in Sf9 cells (D) were determined by Dual-Luciferase® Reporter Assay System as described in the Materials and Methods. (C) The nucleotide sequences of the NF-?B sites from M. sexta moricin (Mor) and lysozyme (Lyz) promoters. The arrows indicate the direction of NF-?B sites. (E) Schematic diagrams of the moricin promoters (see text for detail information). Bars represent the mean of three independent measurements ±SEM.
Fig 3: Activation of moricin promoters by MsFkh and MsRel2-RHD independently. The relative luciferase activities of the truncated Mor-242 and its Fkh-binding site mutant promoters activated by recombinant MsFkh or MsRel2-RHD alone, or by co-expression of MsFkh and MsRel2-RHD in S2 cells were determined by Dual-Luciferase® Reporter Assay System as described in the Materials and Methods. Bars represent the mean of three independent measurements ± SEM. For the activity among different promoters activated by transcription factors (comparing solid bars by MsFkh, stripe bars by MsRel2-RHD, or dotted bars by MsFkh/MsRel2-RHD across the promoters), identical letters (capital letters for solid bars and small letters for stripe bars) or identical numerical numbers (dotted bars) are not significant difference (p > 0.05) while different letters or different numerical numbers indicate significant difference (p < 0.05). Comparing the activity of the same promoter stimulated by different transcription factors (between solid and stripe bars, solid and dotted bars, as well as stripe and dotted bars for each promoter), the significance of difference was also determined by an unpaired t-test (*p < 0.05; **p < 0.01), and “n” indicates not significant.
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