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. 2014 Jan 7;4:487. doi: 10.3389/fimmu.2013.00487

Figure 2.

Figure 2

Purification and functional characterization of the recombinant LT derivatives. (A) SDS-PAGE of the purified recombinant LT (2 μg/lane). Samples: lane 1, LT1; lane 2, LT2; lane 3, LTK63; and MW, PageRuler™ Unstained Protein Ladder (Fermentas). (B) In vitro cAMP accumulation in Y-1 cells exposed to trypsin-activated LT1, LT2, or LTK63 for different time periods. (C) Binding of the purified LTs to Y-1 cells. Cells were incubated with LT1, LT2, or LTK63 for 1 h and labeled with mouse anti-LT and FITC-conjugated anti-mouse IgG antibodies before analysis of emitted fluorescence in a flow cytometer. Samples: LT1 (dotted line); LT2 (black line), and LTK63 (gray line). Untreated Y-1 cells are indicated by the dashed line on the left side of the figure. (D) Receptor binding activities of the purified LT derivatives in GM1-ELISA. Reactions were developed with mouse anti-LT serum and peroxidase-conjugated anti-mouse IgG antibody. (E) Local edema formation in BALB/c mice (n = 5) after i.d. injection of 1 μg of the test LT forms. The experiment was independently performed three times.