CB1954 kinetic experiments were all carried out in a 96-well microtiter plate (Corning, USA) using a Thermoscientific Varioskan 96-well plate microplate reader [29 (link)]. Product formation at 420 nm was measured over time in order to determine the Michaelis–Menten kinetic parameters of CB1954 against the NfnB-Cys:CPP conjugate. CB1954 (0.1–5 mM), NADH (400 µM) and PB (50 mM, pH 7.2) were combined and incubated at 37 °C for 3 min before purified NfnB-Cys or NfnB-Cys:CPP (1:1 ratio) was added (50 μg/mL; again NfnB-Cys:CPP volume added was adjusted to ensure 50 μg/mL of NfnB-Cys was added). Dimethyl sulfoxide (DMSO) solvent concentration was kept constant at 5% v/v to account for any negative solvent related effect [50 (link)]. Hydroxylamine yield per second was determined by calculating the change in absorbance over 20 s and the molar extinction coefficient, which is the same for both products (ε = 1200 M−1cm−1 at 420 nm) [14 (link),18 (link),39 (link),50 (link),51 (link),52 (link),53 (link)]. Data gathered was transferred to SigmaPlot 12 (SPSS, Systat Software Inc.) where a non-linear regression tool was used to generate a Michaelis–Menten hyperbolic curve and a report containing the kinetic information of the system.
Sigmaplot 12
Manufactured by IBM
Sourced in United States
SigmaPlot 12.0 is a data analysis and graphing software developed by Systat Software Inc. It is designed to help users create high-quality scientific graphs and charts. The software offers a range of features, including curve fitting, statistical analysis, and data manipulation tools.
Automatically generated - may contain errors
Lab products found in correlation
Sas 9, by SAS Institute (6 mentions)
Clampfit 10, by Molecular Devices (4 mentions)
Sigmaplot 12, by Merck Group (3 mentions)
Axopatch 200a amplifier, by Molecular Devices (3 mentions)
Clampex version 10, by Molecular Devices (3 mentions)
Statistix 8, by SAS Institute (3 mentions)
96 well plate microplate reader, by Thermo Fisher Scientific (2 mentions)
96 well microtiter plate, by Corning (2 mentions)
Prism 5, by GraphPad (2 mentions)
Multigauge computer software, by Berthold Technologies (1 mentions)
Ecl reagent, by GE Healthcare (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Varioscan 96 well plate microplate reader, by Thermo Fisher Scientific (1 mentions)
Prism 8, by GraphPad (1 mentions)
Spss 22, by IBM (1 mentions)
Instat 3, by GraphPad (1 mentions)
Sigmastat, by IBM (1 mentions)
Originpro 8, by OriginLab (1 mentions)
Spss version 12, by IBM (1 mentions)
Methacholine, by Merck Group (1 mentions)
80 protocols using sigmaplot 12
1
Kinetic Study of CB1954 Reduction by NfnB-Cys:CPP
2
Statistical Analysis of Experimental Data
3
Protein Expression Analysis in APC Mice
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Proteins were extracted from tumours and from adjacent normal tissues of small intestine and colon/rectum of APC∆14/+ mice using the method described previously [5 (link), 14 (link)]. Proteins were separated by running the samples through 10% SDS-PAGE, and then blotted with antibodies against Bmi1 (Gene Tex, Irvine, CA), PAK1 or GAPDH (Cell Signaling, Danvers, MA). Bound antibodies were visualized using ECL reagents (GE Healthcare, Amersham, UK), and the density of each band was analysed using Multi-gauge computer software (Berthold). The correlation of Bmi1 and PAK1 was analysed using the program Sigma Plot 12 (SPSS, Chicago, IL).
4
Cultivar Effects Analysis in Plants
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One-way analysis of variance (ANOVA) and multiple regression analysis were applied to assess the significance of cultivar effect with SAS 9.2 (SAS Institute Inc., USA). Regression analyses between parameters were performed using SigmaPlot 12 (SPSS Inc., Chicago, IL, USA). All regressions were fitted by both linear and power models, and the model with higher regression coefficient was selected.
5
Enzymatic Kinetics Analysis Protocol
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All enzyme kinetics experiments were carried out following the method previously described by Ball et al. (2019 ), using a Thermo Scientific Varioscan 96‐well plate microplate reader. The data were then transferred to SigmaPlot 12 (SPSS, Systat Software Inc.) where a non‐linear regression tool was used to generate a Michaelis–Menten hyperbolic curve and a report containing the important kinetic information of the system under test.
6
Comparative Analysis of Experimental Treatments
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One and two-way analyses of variance (ANOVA) were applied to assess the differences between treatments with Statistics 8.1 analytical software. Linear regression and correlation analysis were performed to test the possible correlations between the studied parameters using Sigma Plot 12 (SPSS Inc., Chicago, IL, USA).
7
Kinetic Analysis of CB1954 Prodrug Reduction
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CB1954 kinetic experiments were all carried out in a 96-well microtiter plate (Corning, USA) using a Thermoscientific Varioskan 96-well plate microplate reader using the method that we have previously described [32 (link),33 (link),34 (link)]. Product formation was measured at 420 nm over time to determine the -Menten kinetic parameters of the CB1954 prodrug with either NfnB-Cys or YfkO-Cys. CB1954 (0.1–10 mM), NADH (400 µM) and PB (50 mM, pH 7.2) were combined and incubated at 37 °C for 3 min before purified NfnB-Cys or YfkO-Cys (10 µg/mL). Hydroxylamine yield per second was determined by calculating the change in absorbance over 20 s and the molar extinction coefficient, which is the same for both products (ε = 1200 M−1 cm−1 at 420 nm) [12 (link),13 (link),15 (link),25 (link),32 (link)]. Data was analysed using SigmaPlot 12 (SPSS, Systat Software Inc., Chicago, USA) where a non-linear regression tool was used to generate a Michaelis-Menten hyperbolic curve and a report containing the important kinetic information of the system under test. Coefficient of Variability (CoV) for the kinetics is 7.06%.
8
Analyzing ORM1 Treatment Effects
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Data were analyzed by analysis of variance using SigmaPlot 12 software (SPSS Science, Chicago, IL) to test for the effects of treatment with ORM1. Mean separation was analyzed using Student-Newman-Keuls test. If Shapiro- Wilk normality test determined the data were not normally distributed, then Kruskal-Wallis one way ANOVA on ranks was performed and all pairwise comparisons were evaluated with Tukey’s test. Means were defined as different at P < 0.05.
9
Analysis of Variance and Regression
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Data were analyzed using one-way analysis of variance (ANOVA) or two-way ANOVA for repeated measures followed by Holm-Sidak multiple comparison tests (SigmaPlot 12, SPSS, Chicago, IL, USA). Regression analyses were performed using SigmaPlot. The threshold value for acceptance of differences was 5%.
10
Statistical Analysis of Experimental Data
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All values in this study are reported as mean plus or minus the standard error of the mean (±s.e.m.). In each case normality of the data distribution was tested and the appropriate parametric or non-parametric tests were used to compare group values using Sigmaplot 12 software (SPSS Inc., Chicago IL). Differences between values were considered to be significant when p < 0.05. Graphs were prepared using either Sigmaplot 12, or Graphpad Prism (v.5). Linear regression analysis was performed using Sigmaplot 12.
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