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Figure 2 | BMC Biotechnology

Figure 2

From: Human TGFalpha-derived peptide TGFalphaL3 fused with superantigen for immunotherapy of EGFR-expressing tumours

Figure 2

TGFαL3SEAD227A promotes splenocyte proliferation and binds to the EGFR. (A) Superantigenic activity of the fusion proteins. 1.0 × 105 freshly isolated splenocytes were seeded in each well in of a 96-well plate at a density of 1.0 × 105 cells/well. The cells were then incubated with 1.0 μg/ml TGFαL3SEAD227A, 1.0 μg/ml SEAD227ATGFαL3, 1.0 μg/ml rSEA, 25 μg/ml PHA or 1.0 μg/ml BSA at 37°C for 72 h, and then MTT solution was dispensed to each well and the optical density at 570 nm was measured. Each column shows the mean ± SD of triplicate samples. (B) The binding of fusion proteins to EGFR. A431 cells were cultured in a 96-well plate, and then fixed with 10% neutral formaldehyde at room temperature for 1 h. The bindings of cells to various concentrations of fusion proteins were detected by cell ELSIA (TGFαL3SEAD227A (black diamond), SEAD227ATGFαL3 (triangle), rSEA (circle). Each point shows the mean ± SD of triplicate samples. (C) Determining the association of proteins to the EGFR via immunofluorescence staining. A431 cells were seeded on a cover glass in a 12-well plate overnight, and then fixed with 10% neutral formaldehyde. The cells were treated with 50 ng/μl rSEA or TGFαL3SEAD227A, and subsequently incubated with anti-hexahistidine mAb and FITC-conjugated rabbit anti-mice IgG. Bar, 8 μm. (D) Binding specificity of TGFαL3SEAD227A to A431 cells. A431 cells were seeded and treated as (C). Then 50 ng/μl EGF, TGFαL3SEAD227A, a mixture of EGF (50 ng/μl) and TGFαL3SEAD227A (50 ng/μl), or PBS were applied to the A431 cells for two hours. After an extensive wash, cells were incubated using anti-EGF, anti-6His, or blank control in PBS (only secondary antibody applied) treated cells. The color was then developed using a standard protocol after the secondary antibody was applied. Bar, 50 μm.

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