Sigmaplot version 11

Data were expressed as the mean ± standard error of the mean (SEM). Statistical analysis was performed with SigmaPlot version 11.0 and SPSS 13.0. Differences between the mean of two groups were assessed by Student's t test, and differences in the mean of multiple groups were assessed by one‐way ANOVA. Correlations of two groups and comparisons of quantitative values of expression were assessed by Pearson's test. A value of P < .05 was considered to indicate a statistically significant difference. All experiments were repeated three times.

Data were expressed as means ± standard errors of the means. Statistical analyses were performed with SigmaPlot version 11.0 and SPSS version 13.0. Differences between means were assessed with the Student's t test, paired t-test, or Mann–Whitney U test in cases of abnormally distributed data. In multiple comparisons, one-way analysis of variance was used. Pearson's test was used to detect the correction of two groups and compare quantitative values of expression. A value of P < 0.05 was considered statistically significant.

We used a microplate enzyme-linked immunosorbent assay (ELISA) for measuring protein-protein interactions [22 (link)], as described in [14 (link)]. Purified DvCad1-CR peptides were biotinylated using sulfo-NHS (N-hydroxysuccinimide)-photocleavable biotin (Pierce, Rockford, IL, USA), dialyzed into 200 mM NaCl, 20 mM Na₂CO₃ (pH 8.0), and stored in aliquots at 4 °C. Microtiter plates (high-binding, 96-well Immulon 2HB plates; Thermo Fisher Scientific, Inc., Waltham, MA) were coated with 1.0 µg Cry3Bb/well in 50 μL coating buffer (100 mM Na₂CO₃, pH 9.6), with or without the 1000-fold molar excess unlabeled DvCad1-CR peptides. In competition binding assays, 20 nM biotinylated DvCad1-CR peptide was mixed with increasing concentrations of the unlabeled DvCad1-CR peptides. Cry3Bb toxin-coated or peptide-coated plates were washed and incubated with horse radish peroxidase-conjugated streptavidin (SA-HRP; Pierce), and then incubated with an HRP chromogenic substrate (1-Step Ultra TMB-ELISA, Thermo Fisher Scientific, Inc., USA), after washing, to detect bound SA-HRP. Color development was stopped by adding 3M sulfuric acid, and absorbance was measured at 450 nm using a microplate reader (MDS Analytical Technologies, Sunnyvale, CA, USA). Data were plotted and analyzed using the Sigma Plot Version 11.0 (SPSS Science, Chicago, IL, USA).