wnt3a growth factor Search Results


recombinant human wnt3a  (Fisher Scientific)
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Fisher Scientific recombinant human wnt3a
RKO cells were treated with 160 ng/mL <t>Wnt3A</t> for the specified times, and then assayed for phospho-LRP5/6 and β-catenin level by Western blot. Error bars are standard error of the mean from 2 to 4 biological replicates. Data are plotted relative to the sample at time zero, and normalized to the average maximal activation across experiments. The grey lines connect the mean of each time point.
Recombinant Human Wnt3a, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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RKO cells were treated with 160 ng/mL Wnt3A for the specified times, and then assayed for phospho-LRP5/6 and β-catenin level by Western blot. Error bars are standard error of the mean from 2 to 4 biological replicates. Data are plotted relative to the sample at time zero, and normalized to the average maximal activation across experiments. The grey lines connect the mean of each time point.

Journal: eLife

Article Title: Signaling pathways as linear transmitters

doi: 10.7554/eLife.33617

Figure Lengend Snippet: RKO cells were treated with 160 ng/mL Wnt3A for the specified times, and then assayed for phospho-LRP5/6 and β-catenin level by Western blot. Error bars are standard error of the mean from 2 to 4 biological replicates. Data are plotted relative to the sample at time zero, and normalized to the average maximal activation across experiments. The grey lines connect the mean of each time point.

Article Snippet: Recombinant human Wnt3A was purchased from Fisher Scientific (5036WN), and recombinant human EGF was purchased from Sigma (E9644).

Techniques: Western Blot, Activation Assay

( A ) Measurements of the input-output relationship in the Wnt pathway. In these experiments, RKO cells were stimulated with 0–1280 ng/mL purified Wnt3A ligand, harvested at 6 hr after ligand stimulation, and lysed for Western blot analyses. Shown on top is a representative Western blot. The data plotted come from seven independent experiments (total N = 66). Each circle indicate the mean intensities of the phospho-LRP5/6 (x-axis) and β-catenin (y-axis) bands for all Western blot biological replicates, and error bars indicate the standard error of the mean. For each gel, we normalize the unstimulated sample (i.e. 0 ng/mL of Wnt3A) to one, and scale the magnitude of the dose response to the average of all gels (described in Materials and methods). The grey line is a least squares regression line, and ρ is the Pearson’s coefficient, where ρ = 1 is a perfect positive linear correlation. ( B ) Measurements of the input-output relationship in the ERK pathway. In these experiments, H1299 cells were stimulated with 0–50 ng/mL purified EGF ligand, harvested at 5 min after ligand stimulation, and lysed for Western blot analyses. Shown on top is a representative Western blot. The data plotted here come from five independent experiments (total N = 30). Each circle indicates the mean intensities of dpERK1/2 bands across Western blot biological replicates, and the error bars indicate standard error of the mean. Single replicates are plotted without error bars. All data is plotted relative to unstimulated sample. The grey line is a least squares regression line, and r 2 is the coefficient of correlation where r 2 = 1 is a perfect linear correlation. ( C ) As in ( A ), except that cells were treated with 1 μM CHIR99021 (detailed in Materials and methods). The data plotted here come from five independent experiments (total N = 59). The grey line is a least squares regression, and ρ is the Pearson’s coefficient, where ρ = 1 is a perfect positive linear correlation. Shown in the subplot are the same least squares regression line (solid line), overlaid with the model prediction (dashed line). ( D ) As in ( B ), but measurements were performed in H1299 cells expressing mutant Raf S289/296/301A. The data plotted here come from three independent experiments (total N = 15). The grey line is a fit using the ERK model. We first fitted the gain of the model to the data (i.e. the y-range), and afterward, varied the strength of dpERK feedback (k 25 ) to find the best fit. We used the weighted Akaike Information Criterion, w(AICc), to verify that the nonlinear fit from the ERK model outperforms a linear least squares fit (see Materials and methods). 0 < w(AICc) < 1, with higher w(AICc) indicates better performance by the non-linear fit. In all figures, linearity was additionally assessed using the least absolute deviations, L1-norm (see Methods). L1-norm can range from 0 to 0.5, with L1-norm < 0.1 indicate a linear relationship. Blue vs grey circles in each figure are explained in the main text. Source files of all Western blot gel images and numerical quantitation data are available in . 10.7554/eLife.33617.022 Figure 3—source data 1.

Journal: eLife

Article Title: Signaling pathways as linear transmitters

doi: 10.7554/eLife.33617

Figure Lengend Snippet: ( A ) Measurements of the input-output relationship in the Wnt pathway. In these experiments, RKO cells were stimulated with 0–1280 ng/mL purified Wnt3A ligand, harvested at 6 hr after ligand stimulation, and lysed for Western blot analyses. Shown on top is a representative Western blot. The data plotted come from seven independent experiments (total N = 66). Each circle indicate the mean intensities of the phospho-LRP5/6 (x-axis) and β-catenin (y-axis) bands for all Western blot biological replicates, and error bars indicate the standard error of the mean. For each gel, we normalize the unstimulated sample (i.e. 0 ng/mL of Wnt3A) to one, and scale the magnitude of the dose response to the average of all gels (described in Materials and methods). The grey line is a least squares regression line, and ρ is the Pearson’s coefficient, where ρ = 1 is a perfect positive linear correlation. ( B ) Measurements of the input-output relationship in the ERK pathway. In these experiments, H1299 cells were stimulated with 0–50 ng/mL purified EGF ligand, harvested at 5 min after ligand stimulation, and lysed for Western blot analyses. Shown on top is a representative Western blot. The data plotted here come from five independent experiments (total N = 30). Each circle indicates the mean intensities of dpERK1/2 bands across Western blot biological replicates, and the error bars indicate standard error of the mean. Single replicates are plotted without error bars. All data is plotted relative to unstimulated sample. The grey line is a least squares regression line, and r 2 is the coefficient of correlation where r 2 = 1 is a perfect linear correlation. ( C ) As in ( A ), except that cells were treated with 1 μM CHIR99021 (detailed in Materials and methods). The data plotted here come from five independent experiments (total N = 59). The grey line is a least squares regression, and ρ is the Pearson’s coefficient, where ρ = 1 is a perfect positive linear correlation. Shown in the subplot are the same least squares regression line (solid line), overlaid with the model prediction (dashed line). ( D ) As in ( B ), but measurements were performed in H1299 cells expressing mutant Raf S289/296/301A. The data plotted here come from three independent experiments (total N = 15). The grey line is a fit using the ERK model. We first fitted the gain of the model to the data (i.e. the y-range), and afterward, varied the strength of dpERK feedback (k 25 ) to find the best fit. We used the weighted Akaike Information Criterion, w(AICc), to verify that the nonlinear fit from the ERK model outperforms a linear least squares fit (see Materials and methods). 0 < w(AICc) < 1, with higher w(AICc) indicates better performance by the non-linear fit. In all figures, linearity was additionally assessed using the least absolute deviations, L1-norm (see Methods). L1-norm can range from 0 to 0.5, with L1-norm < 0.1 indicate a linear relationship. Blue vs grey circles in each figure are explained in the main text. Source files of all Western blot gel images and numerical quantitation data are available in . 10.7554/eLife.33617.022 Figure 3—source data 1.

Article Snippet: Recombinant human Wnt3A was purchased from Fisher Scientific (5036WN), and recombinant human EGF was purchased from Sigma (E9644).

Techniques: Purification, Western Blot, Expressing, Mutagenesis, Quantitation Assay

RKO cells were treated with the specified dose of Wnt3A for six hours, and then assayed for phospho-LRP5/6 and β-catenin by quantitative Western blot. Data are plotted relative to unstimulated samples. ( A–B ) In wt cells, phospho-LRP5/6 ( A ) shows > 90% of maximal response at 200 ng/mL Wnt3A, while β-catenin ( B ) shows 70% of maximal response at 200 ng/mL, and subsequently incremental response until 640 ng/mL Wnt3A. ( C–D ) In cells pre-treated with 1 µM CHIR99021, phospho-LRP5/6 ( C ) shows > 90% of maximal response at 80 ng/mL Wnt3A, while β-catenin ( D ) shows 70% of maximal response at 80 ng/mL and continues incremental activation at higher doses. ( E ). Cells were treated with 160 ng/mL Wnt3A and assayed for phospho-LRP5/6. Cells pre-treated with 1 µM CHIR99021 (N = 3) exhibited 50% the level of phospho-LRP5/6 as untreated cells (N = 3). The grey lines simply connect the means of data.

Journal: eLife

Article Title: Signaling pathways as linear transmitters

doi: 10.7554/eLife.33617

Figure Lengend Snippet: RKO cells were treated with the specified dose of Wnt3A for six hours, and then assayed for phospho-LRP5/6 and β-catenin by quantitative Western blot. Data are plotted relative to unstimulated samples. ( A–B ) In wt cells, phospho-LRP5/6 ( A ) shows > 90% of maximal response at 200 ng/mL Wnt3A, while β-catenin ( B ) shows 70% of maximal response at 200 ng/mL, and subsequently incremental response until 640 ng/mL Wnt3A. ( C–D ) In cells pre-treated with 1 µM CHIR99021, phospho-LRP5/6 ( C ) shows > 90% of maximal response at 80 ng/mL Wnt3A, while β-catenin ( D ) shows 70% of maximal response at 80 ng/mL and continues incremental activation at higher doses. ( E ). Cells were treated with 160 ng/mL Wnt3A and assayed for phospho-LRP5/6. Cells pre-treated with 1 µM CHIR99021 (N = 3) exhibited 50% the level of phospho-LRP5/6 as untreated cells (N = 3). The grey lines simply connect the means of data.

Article Snippet: Recombinant human Wnt3A was purchased from Fisher Scientific (5036WN), and recombinant human EGF was purchased from Sigma (E9644).

Techniques: Western Blot, Activation Assay

In these two independent experiments, RKO cells were stimulated with a range of Wnt3A dose (0-160 ng/mL), the cells lysed after 6 hr, and analyzed for Western blot against β-catenin and phosphorylated LRP5/6 (pLRP5/6). Top row: In each experiment, GAPDH intensity varies with <10% CV across samples. Bottom row: Raw β-catenin and LRP intensity data without normalization with GAPDH loading control. The measurements are plotted relative to unstimulated cells. Grey lines are least squares regression lines, and ρ is the Pearson correlation coefficient.

Journal: eLife

Article Title: Signaling pathways as linear transmitters

doi: 10.7554/eLife.33617

Figure Lengend Snippet: In these two independent experiments, RKO cells were stimulated with a range of Wnt3A dose (0-160 ng/mL), the cells lysed after 6 hr, and analyzed for Western blot against β-catenin and phosphorylated LRP5/6 (pLRP5/6). Top row: In each experiment, GAPDH intensity varies with <10% CV across samples. Bottom row: Raw β-catenin and LRP intensity data without normalization with GAPDH loading control. The measurements are plotted relative to unstimulated cells. Grey lines are least squares regression lines, and ρ is the Pearson correlation coefficient.

Article Snippet: Recombinant human Wnt3A was purchased from Fisher Scientific (5036WN), and recombinant human EGF was purchased from Sigma (E9644).

Techniques: Western Blot, Control