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Fisher Scientific coomassie blue
Effect of site-specific phosphorylation on biochemical characteristics of Pfn1. A) Pull-down assays of indicated GST-tagged Pfn1 constructs with HEK-293 cell lysate were run on an SDS-PAGE and immunoblotted with anti-actin and anti-VASP antibodies (GST was used as a negative control). <t>Coomassie</t> stain in parallel confirms comparable amounts of GST-tagged proteins in the pull-down assay. Note that virtually negligible amount of T89D-Pfn1 was found to be immobilized on glutathione-linked agarose beads. B) Bacteria expressing indicated GST-tagged Pfn1 constructs were lysed with either non-denaturing (containing 1% NP-40) or denaturing (containing 1% NP-40, 2% SDS for one buffer and the other with 6M urea in addition) extraction buffers. Bacterial lysates were immunoblotted with anti-Pfn1 antibody to demonstrate that T89D-Pfn1 is insoluble in non-denaturing lysis buffer. C) HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs were lysed with either non-denaturing (containing 1% NP-40) or denaturing (containing 1% NP-40, 2% SDS for one buffer and the other with 6M urea in addition) extraction buffers. HEK-293 lysates were immunoblotted with anti-GFP antibody to demonstrate that GFP-T89D-Pfn1 is also insoluble in non-denaturing lysis buffer. Note that endogenous Pfn1 level is not affected by expression of any of the ectopic Pfn1 constructs and extractable completely in non-denaturing lysis buffer. D) Fluorescence images of HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs show that EGFP-Pfn1-T89D causes clustering of this fusion protein as indicated by the arrows. Scale bar represents 20 μm. E) HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs were treated with CHX for up to 8 hours. Cell lysates prepared at different time-points after CHX addition were immunoblotted with the indicated antibodies. T89D-Pfn1 undergoes rapid protein degradation while WT- and T89A-Pfn1 are stable over that period of time, similar to the characteristic of endogenous Pfn1 (degradation of p27kip1, a cell-cycle protein that undergoes rapid turnover, validates CHX efficacy). Tubulin blot serves as the loading control. F) The bar graph summarizes quantification of the time-dependent changes in the expression of the indicated GFP-Pfn1 constructs following CHX treatment in HEK-293. Data was summarized from 3 independent experiments (** indicates p
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1) Product Images from "Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A"

Article Title: Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156313

Effect of site-specific phosphorylation on biochemical characteristics of Pfn1. A) Pull-down assays of indicated GST-tagged Pfn1 constructs with HEK-293 cell lysate were run on an SDS-PAGE and immunoblotted with anti-actin and anti-VASP antibodies (GST was used as a negative control). Coomassie stain in parallel confirms comparable amounts of GST-tagged proteins in the pull-down assay. Note that virtually negligible amount of T89D-Pfn1 was found to be immobilized on glutathione-linked agarose beads. B) Bacteria expressing indicated GST-tagged Pfn1 constructs were lysed with either non-denaturing (containing 1% NP-40) or denaturing (containing 1% NP-40, 2% SDS for one buffer and the other with 6M urea in addition) extraction buffers. Bacterial lysates were immunoblotted with anti-Pfn1 antibody to demonstrate that T89D-Pfn1 is insoluble in non-denaturing lysis buffer. C) HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs were lysed with either non-denaturing (containing 1% NP-40) or denaturing (containing 1% NP-40, 2% SDS for one buffer and the other with 6M urea in addition) extraction buffers. HEK-293 lysates were immunoblotted with anti-GFP antibody to demonstrate that GFP-T89D-Pfn1 is also insoluble in non-denaturing lysis buffer. Note that endogenous Pfn1 level is not affected by expression of any of the ectopic Pfn1 constructs and extractable completely in non-denaturing lysis buffer. D) Fluorescence images of HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs show that EGFP-Pfn1-T89D causes clustering of this fusion protein as indicated by the arrows. Scale bar represents 20 μm. E) HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs were treated with CHX for up to 8 hours. Cell lysates prepared at different time-points after CHX addition were immunoblotted with the indicated antibodies. T89D-Pfn1 undergoes rapid protein degradation while WT- and T89A-Pfn1 are stable over that period of time, similar to the characteristic of endogenous Pfn1 (degradation of p27kip1, a cell-cycle protein that undergoes rapid turnover, validates CHX efficacy). Tubulin blot serves as the loading control. F) The bar graph summarizes quantification of the time-dependent changes in the expression of the indicated GFP-Pfn1 constructs following CHX treatment in HEK-293. Data was summarized from 3 independent experiments (** indicates p
Figure Legend Snippet: Effect of site-specific phosphorylation on biochemical characteristics of Pfn1. A) Pull-down assays of indicated GST-tagged Pfn1 constructs with HEK-293 cell lysate were run on an SDS-PAGE and immunoblotted with anti-actin and anti-VASP antibodies (GST was used as a negative control). Coomassie stain in parallel confirms comparable amounts of GST-tagged proteins in the pull-down assay. Note that virtually negligible amount of T89D-Pfn1 was found to be immobilized on glutathione-linked agarose beads. B) Bacteria expressing indicated GST-tagged Pfn1 constructs were lysed with either non-denaturing (containing 1% NP-40) or denaturing (containing 1% NP-40, 2% SDS for one buffer and the other with 6M urea in addition) extraction buffers. Bacterial lysates were immunoblotted with anti-Pfn1 antibody to demonstrate that T89D-Pfn1 is insoluble in non-denaturing lysis buffer. C) HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs were lysed with either non-denaturing (containing 1% NP-40) or denaturing (containing 1% NP-40, 2% SDS for one buffer and the other with 6M urea in addition) extraction buffers. HEK-293 lysates were immunoblotted with anti-GFP antibody to demonstrate that GFP-T89D-Pfn1 is also insoluble in non-denaturing lysis buffer. Note that endogenous Pfn1 level is not affected by expression of any of the ectopic Pfn1 constructs and extractable completely in non-denaturing lysis buffer. D) Fluorescence images of HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs show that EGFP-Pfn1-T89D causes clustering of this fusion protein as indicated by the arrows. Scale bar represents 20 μm. E) HEK-293 cells expressing indicated EGFP-fused Pfn1 constructs were treated with CHX for up to 8 hours. Cell lysates prepared at different time-points after CHX addition were immunoblotted with the indicated antibodies. T89D-Pfn1 undergoes rapid protein degradation while WT- and T89A-Pfn1 are stable over that period of time, similar to the characteristic of endogenous Pfn1 (degradation of p27kip1, a cell-cycle protein that undergoes rapid turnover, validates CHX efficacy). Tubulin blot serves as the loading control. F) The bar graph summarizes quantification of the time-dependent changes in the expression of the indicated GFP-Pfn1 constructs following CHX treatment in HEK-293. Data was summarized from 3 independent experiments (** indicates p

Techniques Used: Construct, SDS Page, Negative Control, Staining, Pull Down Assay, Expressing, Lysis, Fluorescence

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