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. Author manuscript; available in PMC: 2021 Apr 1.
Published in final edited form as: Insect Biochem Mol Biol. 2020 Jan 21;119:103317. doi: 10.1016/j.ibmb.2020.103317

Figure 4. Oligomerization of Cyt1Aa and Cyt1Aa mutants in BBMV or RBC.

Figure 4.

A. Oligomerization of Cyt1Aa and Cyt1Aa mutants with Ae. aegypti BBMV. Soluble Cyt1Aa protoxins were incubated with BBMV and trypsin, the samples were separated by centrifugation and the Cyt1Aa oligomers were detected in the pellets and supernatants after heating the samples (50 °C, 3 min) with loading buffer before loading into SDS-PAGE. The gels were blotted to PVDF membranes and Cyt1Aa oligomers were detected using an anti-Cyt1Aa antibody as indicated in Materials and Methods. Lane 1. Molecular weight marker. Lanes 2 and 3 -Cyt1Aa supernatant (S) or pellet (P) fraction, lanes 4 and 5 - supernatant or pellet fraction of Cyt1AaA65C, lanes 6 and 7 to supernatant or pellet fraction of Cyt1AaL85C, lanes 8 and 9 - supernatant or pellet fraction of Cyt1AaN89C. B. Oligomerization of Cyt1Aa and Cyt1Aa mutants with rabbit RBC. Soluble Cyt1Aa protoxins were incubated with RBC and trypsin, the samples were separated by centrifugation and the Cyt1Aa oligomers were detected in the pellets and supernatants after heating the samples (50 °C, 3 min) with loading buffer before loading into SDS-PAGE. The gels were blotted to PVDF membranes and Cyt1Aa oligomers were detected using an anti-Cyt1Aa antibody as indicated in Materials and Methods. Lane 1. Molecular weight marker. Image shows analysis of pellets (lanes 2, 3, 4 and 5) and supernatant fractions (lanes 6, 7, 8 and 9). Samples of Cyt1Aa (lanes 2 and 6), Cyt1AaA5C (lanes 3 and 7), Cyt1AaL85C (lanes 4 and 8) and Cyt1AaN89C (lanes 5 and 9). Molecular weight markers are indicated in kDa in the left of each panel.