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. 2016 Feb 5;82(4):1023–1034. doi: 10.1128/AEM.02871-15

FIG 4.

FIG 4

Resistance to corn event TC1507 and reduced Cry1Fa toxin binding are associated with reduced levels of alkaline phosphatase transcript and protein levels. (A) Transcript levels for putative Cry1 toxin receptors were quantified and compared between larvae from the Ben and 456 strains using quantitative real-time PCR (qRT-PCR). Shown are the relative transcript levels for S. frugiperda ABCC2 transporter, cadherin, three ALPs, and four APN genes calculated using the ribosomal L18 gene as an internal reference, according to the 2−ΔΔCT method and relative to the transcript levels in Ben larvae (black bar). The data in each bar are the mean with the corresponding standard error calculated from three independent biological samples, each measured in triplicate (n = 3). Asterisks above a column indicate significantly reduced transcripts levels in larvae from the 456 strain compared to those in the Ben strain (ANOVA and post hoc Holm-Šidák test for multiple comparisons to Ben as a control; P < 0.01). (B) Alkaline phosphatase (ALP) protein levels detected in Western blots of BBMV from the Ben, 456F, 456M, and 456 strains. The detection of ALP was accomplished using antisera against A. gambiae mALP, as described in Materials and Methods. Numbers on the left are molecular masses (in kilodaltons). (C) Specific alkaline phosphatase (ALP, gray bars) and aminopeptidase-N (APN, white bars) activity levels in BBMV from the Ben, 456, 456F, and 456M strains, as indicated in the graph. One enzymatic unit was defined as the amount of enzyme that would hydrolyze 1.0 μmol substrate to the chromogenic product per minute at the specific reaction pH and temperature. The data shown are the mean specific activities obtained from the results from at least three independent BBMV batches for each strain measured at least in triplicate. Asterisks above a column indicate significant differences in specific activity for an enzyme among strains (ANOVA and post hoc Holm-Šidák test for multiple pairwise comparisons, P < 0.01). Since APN activity data failed an equal-variance test, in this case, we used ANOVA on ranks (Kruskal-Wallis test, P < 0.01) to determine statistical significance.