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Frap Recovery Data Analysis, supplied by GraphPad Software Inc, 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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frap assay data analysis  (GraphPad Software Inc)
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APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by <t>FRAP</t> in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad <t>Prism</t> <t>software</t> as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).
Frap Assay Data Analysis, supplied by GraphPad Software Inc, 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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frap data analysis software graphpad prism 9  (GraphPad Software Inc)
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APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by <t>FRAP</t> in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad <t>Prism</t> <t>software</t> as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).
Frap Data Analysis Software Graphpad Prism 9, supplied by GraphPad Software Inc, 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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frap data analysis  (Inserm Transfert)
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Inserm Transfert frap data analysis
APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by <t>FRAP</t> in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad <t>Prism</t> <t>software</t> as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).
Frap Data Analysis, supplied by Inserm Transfert, 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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APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by FRAP in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad Prism software as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).

Journal: Cancers

Article Title: Characterization of Adenomatous Polyposis Coli Protein Dynamics and Localization at the Centrosome

doi: 10.3390/cancers8050047

Figure Lengend Snippet: APC full-length and mutant dynamics at the centrosome are slowed by nocodazole treatment. ( A ) pAPC-FL-GFP and pAPC1-1309-GFP ( green ) were each co-transfected with pRFP-PCNT-C241 (red) into HeLa cells. APC-GFP was analysed for dynamic recruitment at the centrosome by FRAP in the presence and absence of 33 µM nocodazole. The effect of γ-tubulin on APC dynamics was also tested where FRAP was performed after depletion with γ-tubulin siRNA. ( B ) Fluorescence recovery curves were plotted as shown for APC-FL, indicating relative rates of recovery and equilibration (plateau) at the centrosome for up to 100 s after bleaching. The presence of nocodazole ( black dashed line ) significantly reduced the rate of recovery of APC-FL-GFP compared to that of untreated cells ( blue line ) ( n = 20–30). This was also indicated by comparison of T 1/2 values for the fast recovery pools (T = 0–40 s) ( p < 0.0001), and extrapolated retention levels, calculated from the recovery curve data using Graph Pad Prism software as above (see ). ( C ) The dynamic exchange profile of APC1-1309 at the centrosome +/− nocodazole ( black dotted line ) showed a small difference in the dynamic rate of recruitment compared to untreated cells ( p = 0.0447). There was a small but significant difference in T 1/2 value; however, no change in retention after nocodazole treatment. ( D ) Fluorescence recovery curves are shown for APC1-1309 for siCTRL ( red ) and γ-tubulin siRNA ( green ) transfected cells ( n = 9–10). Confirmation of γ-tubulin knockdown was by Western blot, and vinculin was used as loading control. Column graph shows the T 1/2 of the fluorescence recovery over 40 s, which was significantly increased after the knockdown of γ-tubulin ( p = 0.0194). No significant change in the maximum recovery (retention) was detected. (*, p < 0.05; ****, p < 0.0001).

Article Snippet: The FRAP assay data was analysed in GraphPad Prism 5 (GraphPad Software Inc., La Jolla, CA, USA) to determine the rate of fluorescence recovery over time, and the relative size of the dynamic and immobile pools of APC ( B–D).

Techniques: Mutagenesis, Transfection, Fluorescence, Comparison, Software, Knockdown, Western Blot, Control