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inactive analogue pp3  (Millipore)


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    Structured Review

    Millipore inactive analogue pp3
    ( A–B ) To analyze the impact of Src kinase inhibition on actin dynamics, ACCs were permeabilized in the presence of Alexa Fluor 488 G actin, 10 µM free Ca 2+ and 10 µM of the Src inhibitor PP2 or its inactive analogue <t>PP3.</t> ( C–D ) To evaluate the drug effects on the organization of the cortical actin network, cells were permeabilized with digitonin for 6 minutes in the presence of PP2 (or PP3), then fixed and stained with 1 µM phalloidin-rhodamine B. ( E–H ) Intact cells were incubated for 20 min with 10 µM PP2 (or PP3), maintained in resting condition (E–F) or stimulated with 20 µM ionomycin for 10 s (G–H), then fixed and stained with 1 µM phalloidin-rhodamine B to confocal visualization. A, C, E and G show representative confocal images for each condition. Scale bar = 10 µm. B, D, F and H correspond to the quantification of the cortical actin fluorescence intensity, where data are means ± SEM from at least 16 cells per each condition (*p<0.05 compared with PP3). Note that Src kinases inhibition with PP2 reduces the new formation of cortical actin filaments, but it does not affect the cortical actin network in permeabilized or intact cells.
    Inactive Analogue Pp3, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/inactive+analogue+pp3/inactive+analogue+pp3/pmc04047038-35-26-29
    Average 90 stars, based on 1 article reviews
    inactive analogue pp3 - by Bioz Stars, 2026-11
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    Images

    1) Product Images from "Src Kinases Regulate De Novo Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells"

    Article Title: Src Kinases Regulate De Novo Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0099001

    ( A–B ) To analyze the impact of Src kinase inhibition on actin dynamics, ACCs were permeabilized in the presence of Alexa Fluor 488 G actin, 10 µM free Ca 2+ and 10 µM of the Src inhibitor PP2 or its inactive analogue PP3. ( C–D ) To evaluate the drug effects on the organization of the cortical actin network, cells were permeabilized with digitonin for 6 minutes in the presence of PP2 (or PP3), then fixed and stained with 1 µM phalloidin-rhodamine B. ( E–H ) Intact cells were incubated for 20 min with 10 µM PP2 (or PP3), maintained in resting condition (E–F) or stimulated with 20 µM ionomycin for 10 s (G–H), then fixed and stained with 1 µM phalloidin-rhodamine B to confocal visualization. A, C, E and G show representative confocal images for each condition. Scale bar = 10 µm. B, D, F and H correspond to the quantification of the cortical actin fluorescence intensity, where data are means ± SEM from at least 16 cells per each condition (*p<0.05 compared with PP3). Note that Src kinases inhibition with PP2 reduces the new formation of cortical actin filaments, but it does not affect the cortical actin network in permeabilized or intact cells.
    Figure Legend Snippet: ( A–B ) To analyze the impact of Src kinase inhibition on actin dynamics, ACCs were permeabilized in the presence of Alexa Fluor 488 G actin, 10 µM free Ca 2+ and 10 µM of the Src inhibitor PP2 or its inactive analogue PP3. ( C–D ) To evaluate the drug effects on the organization of the cortical actin network, cells were permeabilized with digitonin for 6 minutes in the presence of PP2 (or PP3), then fixed and stained with 1 µM phalloidin-rhodamine B. ( E–H ) Intact cells were incubated for 20 min with 10 µM PP2 (or PP3), maintained in resting condition (E–F) or stimulated with 20 µM ionomycin for 10 s (G–H), then fixed and stained with 1 µM phalloidin-rhodamine B to confocal visualization. A, C, E and G show representative confocal images for each condition. Scale bar = 10 µm. B, D, F and H correspond to the quantification of the cortical actin fluorescence intensity, where data are means ± SEM from at least 16 cells per each condition (*p<0.05 compared with PP3). Note that Src kinases inhibition with PP2 reduces the new formation of cortical actin filaments, but it does not affect the cortical actin network in permeabilized or intact cells.

    Techniques Used: Inhibition, Staining, Incubation, Fluorescence

    ACCs were incubated with the Src kinase inhibitor PP2 (10 µM) or its inactive isomer PP3 for 20 min, or injected with 5 µM GST, or c-Src SH2-GST (SH2) or cSrc SH3-GST (SH3) domain. Exocytosis was induced with the Ca +2 ionophore ionomycin (20 µM) and monitored by amperometry. ( A ) A representative amperometric trace from a non-treated cell (Control). ( B–C ) Cumulative histograms of the number of amperometric events from non-treated cells (control), or cells treated with PP2 or PP3, or injected with GST, SH2 or SH3. Data are means ± SEM from 12–20 cells. *p<0.05 compared with PP3.
    Figure Legend Snippet: ACCs were incubated with the Src kinase inhibitor PP2 (10 µM) or its inactive isomer PP3 for 20 min, or injected with 5 µM GST, or c-Src SH2-GST (SH2) or cSrc SH3-GST (SH3) domain. Exocytosis was induced with the Ca +2 ionophore ionomycin (20 µM) and monitored by amperometry. ( A ) A representative amperometric trace from a non-treated cell (Control). ( B–C ) Cumulative histograms of the number of amperometric events from non-treated cells (control), or cells treated with PP2 or PP3, or injected with GST, SH2 or SH3. Data are means ± SEM from 12–20 cells. *p<0.05 compared with PP3.

    Techniques Used: Incubation, Injection

    Exocytosis was induced with 20 µM ionomycin and monitored by amperometry. Cells were incubated with 10 µM PP2 or its inactive isomer PP3 for 20 min before the exocytosis induction. These agents were present during the recording. GST, c-Src SH2-GST (SH2) or c-Src SH3-GST (SH3) was injected 30 min before cell stimulation. ( A ) Scheme of an amperometric spike with the analyzed parameters: peak amplitude (Imax), quantal size (Q), half-width (t 1/2 ), rise time (tP) and food duration. ( B ) Representative amperometric spikes from cells treated with PP3 or PP2, or injected with GST, SH2 or SH3. ( C ) Data show average values ± S.E.M. of Imax, Q, t 1/2 , tP, foot frequency and foot duration of amperometric events in control cells (n = 35) or cells treated with PP3 (n = 15), PP2 (n = 20) or injected with GST (n = 13), SH2 (n = 12), SH3 (n = 15). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. & p<0.05 compared with control; *p<0.05 compared with PP3; † p<0.05 compared with GST.
    Figure Legend Snippet: Exocytosis was induced with 20 µM ionomycin and monitored by amperometry. Cells were incubated with 10 µM PP2 or its inactive isomer PP3 for 20 min before the exocytosis induction. These agents were present during the recording. GST, c-Src SH2-GST (SH2) or c-Src SH3-GST (SH3) was injected 30 min before cell stimulation. ( A ) Scheme of an amperometric spike with the analyzed parameters: peak amplitude (Imax), quantal size (Q), half-width (t 1/2 ), rise time (tP) and food duration. ( B ) Representative amperometric spikes from cells treated with PP3 or PP2, or injected with GST, SH2 or SH3. ( C ) Data show average values ± S.E.M. of Imax, Q, t 1/2 , tP, foot frequency and foot duration of amperometric events in control cells (n = 35) or cells treated with PP3 (n = 15), PP2 (n = 20) or injected with GST (n = 13), SH2 (n = 12), SH3 (n = 15). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. & p<0.05 compared with control; *p<0.05 compared with PP3; † p<0.05 compared with GST.

    Techniques Used: Incubation, Injection, Cell Stimulation

    Related Articles

    Incubation:

    Article Title: Lamellipodia mediate the heterogeneity of central olfactory ensheathing cell interactions.
    Article Snippet: The growth and guidance of primary olfactory axons are partly attributed to the presence of olfactory ensheathing cells (OECs).. However, little is understood about the differences between the subpopulations of OECs and what regulates their interactions.. We used OEC-axon assays and determined that axons respond differently to peripheral and central OECs.

    Article Title: Lamellipodia mediate the heterogeneity of central olfactory ensheathing cell interactions
    Article Snippet: .. OECs were incubated with each of the following: recombinant rat glial cell line-derived neurotrophic factor (GDNF; Bio-Scientific, NSW, Australia), nerve growth factor (NGF; Invitrogen Corporation, Melbourne, Australia) at final concentrations of 10 ng/ml and 20 ng/ml; JNK inhibitor II SP600125 (50 nM; Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany), the inactive analogue PP3 (5 μM; Calbiochem), Rac 1 inhibitor (NSC23766, 5, 10 μM; Calbiochem), and Mek 1 inhibitor (ethanolate U0126; 5, 10, 20 μM; Sigma–Aldrich). ..

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Article Title: Src Kinases Regulate De Novo Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells
    Article Snippet: .. To evaluate the role of Src kinases, cells were incubated during 20 minutes at 37°C with 10 μM of the Src kinase inhibitor PP2 or its inactive analogue PP3 (Sigma Aldrich). .. To acutely inhibit SH2 or SH3 domain-dependent Src kinase association to different partners, cultured ACCs were injected with 5 μM of SH2 or SH3-GST fusion proteins (or GST alone), using an InjectMan system (Eppendorf) and 0.5 μm- diameter femtotips (Eppendorf) and 30 minutes later monitored by amperometry.

    Article Title: Motile membrane protrusions regulate cell-cell adhesion and migration of olfactory ensheathing glia.
    Article Snippet: Migration of OECs from five points on both sides of the gap over a 6-h period was imaged and measured using the calliper tool in MetaMorph analysis software. .. OECs were incubated with each of the following: recombinant rat GDNF (Bio-Scientific, NSW, Australia), GLIA DOI 10.1002/glia nerve growth factor (NGF, Invitrogen Corporation, Melbourne, Australia) at final concentrations of 5 ng/mL and 10 ng/mL, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 lM; Calbiochem, Merck, Darmstadt, Germany), the inactive analogue PP3 (5 lM; Calbiochem), Rac 1 inhibitor (NSC23766, 100, 1, 5, and 10 lM Calbiochem), Mek 1 inhibitor (Ethanolate U0126, 5, 10, and 20 lM SigmaAldrich), AKT 1/2 inhibitor (AKT 1/2 kinase, 10, 20, 50, and 100 lM, Sigma-Aldrich), PI3K inhibitor (LY294002, 5, 10, 20, and 50 lM Sigma-Aldrich), and Rock inhibitor (Y27632 Dihydrochloride, 5, 10, 20, and 50 lM SigmaAldrich). ..

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Growth factor and inhibitor assays of OE explants Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Recombinant:

    Article Title: Lamellipodia mediate the heterogeneity of central olfactory ensheathing cell interactions.
    Article Snippet: The growth and guidance of primary olfactory axons are partly attributed to the presence of olfactory ensheathing cells (OECs).. However, little is understood about the differences between the subpopulations of OECs and what regulates their interactions.. We used OEC-axon assays and determined that axons respond differently to peripheral and central OECs.

    Article Title: Lamellipodia mediate the heterogeneity of central olfactory ensheathing cell interactions
    Article Snippet: .. OECs were incubated with each of the following: recombinant rat glial cell line-derived neurotrophic factor (GDNF; Bio-Scientific, NSW, Australia), nerve growth factor (NGF; Invitrogen Corporation, Melbourne, Australia) at final concentrations of 10 ng/ml and 20 ng/ml; JNK inhibitor II SP600125 (50 nM; Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany), the inactive analogue PP3 (5 μM; Calbiochem), Rac 1 inhibitor (NSC23766, 5, 10 μM; Calbiochem), and Mek 1 inhibitor (ethanolate U0126; 5, 10, 20 μM; Sigma–Aldrich). ..

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Article Title: Motile membrane protrusions regulate cell-cell adhesion and migration of olfactory ensheathing glia.
    Article Snippet: Migration of OECs from five points on both sides of the gap over a 6-h period was imaged and measured using the calliper tool in MetaMorph analysis software. .. OECs were incubated with each of the following: recombinant rat GDNF (Bio-Scientific, NSW, Australia), GLIA DOI 10.1002/glia nerve growth factor (NGF, Invitrogen Corporation, Melbourne, Australia) at final concentrations of 5 ng/mL and 10 ng/mL, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 lM; Calbiochem, Merck, Darmstadt, Germany), the inactive analogue PP3 (5 lM; Calbiochem), Rac 1 inhibitor (NSC23766, 100, 1, 5, and 10 lM Calbiochem), Mek 1 inhibitor (Ethanolate U0126, 5, 10, and 20 lM SigmaAldrich), AKT 1/2 inhibitor (AKT 1/2 kinase, 10, 20, 50, and 100 lM, Sigma-Aldrich), PI3K inhibitor (LY294002, 5, 10, 20, and 50 lM Sigma-Aldrich), and Rock inhibitor (Y27632 Dihydrochloride, 5, 10, 20, and 50 lM SigmaAldrich). ..

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Growth factor and inhibitor assays of OE explants Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Infection:

    Article Title: Role of Herpes Simplex Virus VP11/12 Tyrosine-Based Motifs in Binding and Activation of the Src Family Kinase Lck and Recruitment of p85, Grb2, and Shc
    Article Snippet: .. Jurkat E6-1 cells were infected in serum-free RPMI 1640 supplemented with the SFK inhibitor PP2 or the inactive analogue PP3 (10 μM; both from Calbiochem) for 1 h. Cells were then maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and either PP2 or PP3 (10 μM). ..

    Olfactory:

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Growth factor and inhibitor assays of OE explants Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Transgenic Assay:

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Article Title: Stimulation of olfactory ensheathing cell motility enhances olfactory axon growth
    Article Snippet: .. Growth factor and inhibitor assays of OE explants Olfactory epithelium (OE) from E11.5–E14.5 OMP-ZsGreen × S100ß-DsRed transgenic embryos were grown for 24 h. OE explants were imaged 20 min prior to the incubation, and then each of the following were added to the culture medium: recombinant human glial cell line-derived neurotrophic factor (GDNF, Bio-Scientific, NSW, Australia), nerve growth factor (NGF, Invitrogen, Melbourne, Australia) at final concentrations of 10 and 20 ng/ml, JNK inhibitor II SP600125 (50 nM, Sigma-Aldrich), Src family kinase inhibitor PP2 (5 μM; Calbiochem, Merck, Darmstadt, Germany) and the inactive analogue PP3 (5 μM; Calbiochem). ..

    Saline:

    Article Title: Activation of Src family kinases in spinal microglia contributes to formalin-induced persistent pain state through p38 pathway.
    Article Snippet: 1008 Abstract: Protein tyrosine phosphorylation has been implicated in normal and pathological functions such as cell proliferation, migration and differentiation.. Recently, some studies have shown that Src family kinases (SFKs) were involved in neurological disorders and neuropathic pain states in which microglial activation plays a role.. In the formalin test, we have reported that microglia undergo at least 2 distinct stages of activation on the basis of signaling events regarding p38 mitogenactivated protein kinases (MAPK).



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    Millipore inactive analogue for src inhibitor pp3
    ( a ) Western blot analysis of SFK activation in HUVEC in response to TNF-α stimulation (10 ng ml −1 ). A phospho-specific antibody against Y416 in SFKs (pY416SFK) was used as an indicator of SFK activation. SFK activation was blocked with the pharmacological antagonist PP2 (10 μM) but not by its inactive analogue <t>PP3</t> (10 μM). Antibody specificity for the phosphorylated form of the kinases was confirmed by dephosphorylating proteins in cell lysates with alkaline phosphatase. * P <0.05 as compared with unstimulated control (lane 1) and # P <0.001 as compared with lane 1 ( n =3). ( b ) TNF-α-induced ATP release from HUVEC following SFK inhibition with PP2. * P <0.05 versus control and PP3 treatments ( n =5). ( c ) Topological schematic of Panx1 highlighting an epitope in the intracellular loop which contains tyrosine 198. This epitope was used to develop antibodies specific to the phosphorylated (pY198Panx1) and non-phosphorylated (Panx1-IL) forms of the protein. ( d ) Overlay of pY198Panx1 signal and Panx1-IL (total) signal as assessed by western blotting with LiCOR IRDye secondary antibodies. pY198Panx1 detects a single species at ∼55 kDa. ( e ) Western blot analysis of pY198Panx1 in HUVEC transfected with plasmids encoding c-Src and/or inhibitor of Src (i-Src). ( f ) Western blot analysis of Panx1 phosphorylation at Y198 in HUVECs stimulated with TNF-α (10 ng ml −1 ). Phospho-signal was normalized to total Panx1 expression using the Panx1-IL Ab. * P <0.05 compared with vehicle control (lane 1) and # P <0.01 compared with 5-min TNF-α stimulation (lane 4) ( n =3). ( g ) TNF-α-induced ATP release from mesenteric venules treated with PP2 (10 μM) or PP3 (10 μM). * P <0.05 as compared with vehicle control ( n =5). ( h , i ) Western blot analysis of TNF-α-induced SFK activation ( h ) and pY198Panx1 phosphorylation ( i ) in isolated mesenteric venules perfused with TNF-α (50 ng ml −1 ) for 30 min. ( j ) Immunofluorescence micrographs of pY198Panx1 in isolated mesenteric venule cross-sections. Venules were isolated from mice expressing endogenous Panx1 in the vascular wall ( VECadER T2+ /Panx1 fl/fl +peanut oil) or mice with specific EC Panx1 deletion ( VECadER T2+ /Panx1 fl/fl +tamoxifen) and stimulated with TNF-α. Asterisks indicate the vessel lumen and nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 30 μm. All data are presented as mean±s.e.m. (error bars). Statistical analyses were performed using one-way analysis of variance.
    Inactive Analogue For Src Inhibitor Pp3, supplied by Millipore, 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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    ( A–B ) To analyze the impact of Src kinase inhibition on actin dynamics, ACCs were permeabilized in the presence of Alexa Fluor 488 G actin, 10 µM free Ca 2+ and 10 µM of the Src inhibitor PP2 or its inactive analogue PP3. ( C–D ) To evaluate the drug effects on the organization of the cortical actin network, cells were permeabilized with digitonin for 6 minutes in the presence of PP2 (or PP3), then fixed and stained with 1 µM phalloidin-rhodamine B. ( E–H ) Intact cells were incubated for 20 min with 10 µM PP2 (or PP3), maintained in resting condition (E–F) or stimulated with 20 µM ionomycin for 10 s (G–H), then fixed and stained with 1 µM phalloidin-rhodamine B to confocal visualization. A, C, E and G show representative confocal images for each condition. Scale bar = 10 µm. B, D, F and H correspond to the quantification of the cortical actin fluorescence intensity, where data are means ± SEM from at least 16 cells per each condition (*p<0.05 compared with PP3). Note that Src kinases inhibition with PP2 reduces the new formation of cortical actin filaments, but it does not affect the cortical actin network in permeabilized or intact cells.

    Journal: PLoS ONE

    Article Title: Src Kinases Regulate De Novo Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells

    doi: 10.1371/journal.pone.0099001

    Figure Lengend Snippet: ( A–B ) To analyze the impact of Src kinase inhibition on actin dynamics, ACCs were permeabilized in the presence of Alexa Fluor 488 G actin, 10 µM free Ca 2+ and 10 µM of the Src inhibitor PP2 or its inactive analogue PP3. ( C–D ) To evaluate the drug effects on the organization of the cortical actin network, cells were permeabilized with digitonin for 6 minutes in the presence of PP2 (or PP3), then fixed and stained with 1 µM phalloidin-rhodamine B. ( E–H ) Intact cells were incubated for 20 min with 10 µM PP2 (or PP3), maintained in resting condition (E–F) or stimulated with 20 µM ionomycin for 10 s (G–H), then fixed and stained with 1 µM phalloidin-rhodamine B to confocal visualization. A, C, E and G show representative confocal images for each condition. Scale bar = 10 µm. B, D, F and H correspond to the quantification of the cortical actin fluorescence intensity, where data are means ± SEM from at least 16 cells per each condition (*p<0.05 compared with PP3). Note that Src kinases inhibition with PP2 reduces the new formation of cortical actin filaments, but it does not affect the cortical actin network in permeabilized or intact cells.

    Article Snippet: To evaluate the role of Src kinases, cells were incubated during 20 minutes at 37°C with 10 µM of the Src kinase inhibitor PP2 or its inactive analogue PP3 (Sigma Aldrich).

    Techniques: Inhibition, Staining, Incubation, Fluorescence

    ACCs were incubated with the Src kinase inhibitor PP2 (10 µM) or its inactive isomer PP3 for 20 min, or injected with 5 µM GST, or c-Src SH2-GST (SH2) or cSrc SH3-GST (SH3) domain. Exocytosis was induced with the Ca +2 ionophore ionomycin (20 µM) and monitored by amperometry. ( A ) A representative amperometric trace from a non-treated cell (Control). ( B–C ) Cumulative histograms of the number of amperometric events from non-treated cells (control), or cells treated with PP2 or PP3, or injected with GST, SH2 or SH3. Data are means ± SEM from 12–20 cells. *p<0.05 compared with PP3.

    Journal: PLoS ONE

    Article Title: Src Kinases Regulate De Novo Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells

    doi: 10.1371/journal.pone.0099001

    Figure Lengend Snippet: ACCs were incubated with the Src kinase inhibitor PP2 (10 µM) or its inactive isomer PP3 for 20 min, or injected with 5 µM GST, or c-Src SH2-GST (SH2) or cSrc SH3-GST (SH3) domain. Exocytosis was induced with the Ca +2 ionophore ionomycin (20 µM) and monitored by amperometry. ( A ) A representative amperometric trace from a non-treated cell (Control). ( B–C ) Cumulative histograms of the number of amperometric events from non-treated cells (control), or cells treated with PP2 or PP3, or injected with GST, SH2 or SH3. Data are means ± SEM from 12–20 cells. *p<0.05 compared with PP3.

    Article Snippet: To evaluate the role of Src kinases, cells were incubated during 20 minutes at 37°C with 10 µM of the Src kinase inhibitor PP2 or its inactive analogue PP3 (Sigma Aldrich).

    Techniques: Incubation, Injection

    Exocytosis was induced with 20 µM ionomycin and monitored by amperometry. Cells were incubated with 10 µM PP2 or its inactive isomer PP3 for 20 min before the exocytosis induction. These agents were present during the recording. GST, c-Src SH2-GST (SH2) or c-Src SH3-GST (SH3) was injected 30 min before cell stimulation. ( A ) Scheme of an amperometric spike with the analyzed parameters: peak amplitude (Imax), quantal size (Q), half-width (t 1/2 ), rise time (tP) and food duration. ( B ) Representative amperometric spikes from cells treated with PP3 or PP2, or injected with GST, SH2 or SH3. ( C ) Data show average values ± S.E.M. of Imax, Q, t 1/2 , tP, foot frequency and foot duration of amperometric events in control cells (n = 35) or cells treated with PP3 (n = 15), PP2 (n = 20) or injected with GST (n = 13), SH2 (n = 12), SH3 (n = 15). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. & p<0.05 compared with control; *p<0.05 compared with PP3; † p<0.05 compared with GST.

    Journal: PLoS ONE

    Article Title: Src Kinases Regulate De Novo Actin Polymerization during Exocytosis in Neuroendocrine Chromaffin Cells

    doi: 10.1371/journal.pone.0099001

    Figure Lengend Snippet: Exocytosis was induced with 20 µM ionomycin and monitored by amperometry. Cells were incubated with 10 µM PP2 or its inactive isomer PP3 for 20 min before the exocytosis induction. These agents were present during the recording. GST, c-Src SH2-GST (SH2) or c-Src SH3-GST (SH3) was injected 30 min before cell stimulation. ( A ) Scheme of an amperometric spike with the analyzed parameters: peak amplitude (Imax), quantal size (Q), half-width (t 1/2 ), rise time (tP) and food duration. ( B ) Representative amperometric spikes from cells treated with PP3 or PP2, or injected with GST, SH2 or SH3. ( C ) Data show average values ± S.E.M. of Imax, Q, t 1/2 , tP, foot frequency and foot duration of amperometric events in control cells (n = 35) or cells treated with PP3 (n = 15), PP2 (n = 20) or injected with GST (n = 13), SH2 (n = 12), SH3 (n = 15). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. & p<0.05 compared with control; *p<0.05 compared with PP3; † p<0.05 compared with GST.

    Article Snippet: To evaluate the role of Src kinases, cells were incubated during 20 minutes at 37°C with 10 µM of the Src kinase inhibitor PP2 or its inactive analogue PP3 (Sigma Aldrich).

    Techniques: Incubation, Injection, Cell Stimulation

    A. UM-SCC47 cells were treated for 72 hr with either control siRNA, or siRNA specific for human Sdc4, β4 integrin (ITGB4), EGFR, or α3 integrin (ITGA3), followed by addition of EdU for 45 min to monitor DNA synthesis prior to fixation and staining; Western blots showing individual receptor expression 72 hr after siRNA transfection are shown (inset); B . Either NOKs or UM-SCC47 cells were allowed to invade through LN332-coated filters for 16 hr in serum-free conditions following stimulation with 10 ng/ml EGF +/− 30 μM SSTN EGFR or vehicle; C. Quantification of NOK or UM-SCC47 cell invasion as in ( B ) in the presence or absence of the EGFR kinase inhibitors gefitinib (3 μM) or erlotinib (2 μM), SFK inhibitor PP2 or its inactive analogue PP3 (3 μM), MST1R/RON inhibitors CAS 913376-84-8 (1 μM) or BMS-0777607 (3 μM), c-Abl inhibitor GNF5 (2 μM), pan-p38MAPK inhibitor BIRB-796 (100 nM) and α6β4 (3E1) or α3β1 (P1B5) integrin blocking antibody (10 μg/ml); D. DNA synthesis is detected by EdU incorporation in UM-SCC47 cells grown for 3 hr in medium containing SSTN EGFR , the EGFR kinase inhibitors gefitinib or erlotinib, or SFK inhibitor PP2 versus its inactive analogue PP3.

    Journal: bioRxiv

    Article Title: MST1R/RON and EGFR in a complex with syndecans sustain carcinoma S-phase progression by preventing p38MAPK activation

    doi: 10.1101/252742

    Figure Lengend Snippet: A. UM-SCC47 cells were treated for 72 hr with either control siRNA, or siRNA specific for human Sdc4, β4 integrin (ITGB4), EGFR, or α3 integrin (ITGA3), followed by addition of EdU for 45 min to monitor DNA synthesis prior to fixation and staining; Western blots showing individual receptor expression 72 hr after siRNA transfection are shown (inset); B . Either NOKs or UM-SCC47 cells were allowed to invade through LN332-coated filters for 16 hr in serum-free conditions following stimulation with 10 ng/ml EGF +/− 30 μM SSTN EGFR or vehicle; C. Quantification of NOK or UM-SCC47 cell invasion as in ( B ) in the presence or absence of the EGFR kinase inhibitors gefitinib (3 μM) or erlotinib (2 μM), SFK inhibitor PP2 or its inactive analogue PP3 (3 μM), MST1R/RON inhibitors CAS 913376-84-8 (1 μM) or BMS-0777607 (3 μM), c-Abl inhibitor GNF5 (2 μM), pan-p38MAPK inhibitor BIRB-796 (100 nM) and α6β4 (3E1) or α3β1 (P1B5) integrin blocking antibody (10 μg/ml); D. DNA synthesis is detected by EdU incorporation in UM-SCC47 cells grown for 3 hr in medium containing SSTN EGFR , the EGFR kinase inhibitors gefitinib or erlotinib, or SFK inhibitor PP2 versus its inactive analogue PP3.

    Article Snippet: SFK inhibitor (PP2) and its paired inactive analogue (PP3), as well as the MST1R/RON inhibitor CAS 913376-84-8 were from Millipore-Sigma.

    Techniques: DNA Synthesis, Staining, Western Blot, Expressing, Transfection, Blocking Assay

    ( a ) Western blot analysis of SFK activation in HUVEC in response to TNF-α stimulation (10 ng ml −1 ). A phospho-specific antibody against Y416 in SFKs (pY416SFK) was used as an indicator of SFK activation. SFK activation was blocked with the pharmacological antagonist PP2 (10 μM) but not by its inactive analogue PP3 (10 μM). Antibody specificity for the phosphorylated form of the kinases was confirmed by dephosphorylating proteins in cell lysates with alkaline phosphatase. * P <0.05 as compared with unstimulated control (lane 1) and # P <0.001 as compared with lane 1 ( n =3). ( b ) TNF-α-induced ATP release from HUVEC following SFK inhibition with PP2. * P <0.05 versus control and PP3 treatments ( n =5). ( c ) Topological schematic of Panx1 highlighting an epitope in the intracellular loop which contains tyrosine 198. This epitope was used to develop antibodies specific to the phosphorylated (pY198Panx1) and non-phosphorylated (Panx1-IL) forms of the protein. ( d ) Overlay of pY198Panx1 signal and Panx1-IL (total) signal as assessed by western blotting with LiCOR IRDye secondary antibodies. pY198Panx1 detects a single species at ∼55 kDa. ( e ) Western blot analysis of pY198Panx1 in HUVEC transfected with plasmids encoding c-Src and/or inhibitor of Src (i-Src). ( f ) Western blot analysis of Panx1 phosphorylation at Y198 in HUVECs stimulated with TNF-α (10 ng ml −1 ). Phospho-signal was normalized to total Panx1 expression using the Panx1-IL Ab. * P <0.05 compared with vehicle control (lane 1) and # P <0.01 compared with 5-min TNF-α stimulation (lane 4) ( n =3). ( g ) TNF-α-induced ATP release from mesenteric venules treated with PP2 (10 μM) or PP3 (10 μM). * P <0.05 as compared with vehicle control ( n =5). ( h , i ) Western blot analysis of TNF-α-induced SFK activation ( h ) and pY198Panx1 phosphorylation ( i ) in isolated mesenteric venules perfused with TNF-α (50 ng ml −1 ) for 30 min. ( j ) Immunofluorescence micrographs of pY198Panx1 in isolated mesenteric venule cross-sections. Venules were isolated from mice expressing endogenous Panx1 in the vascular wall ( VECadER T2+ /Panx1 fl/fl +peanut oil) or mice with specific EC Panx1 deletion ( VECadER T2+ /Panx1 fl/fl +tamoxifen) and stimulated with TNF-α. Asterisks indicate the vessel lumen and nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 30 μm. All data are presented as mean±s.e.m. (error bars). Statistical analyses were performed using one-way analysis of variance.

    Journal: Nature Communications

    Article Title: Pannexin 1 channels regulate leukocyte emigration through the venous endothelium during acute inflammation

    doi: 10.1038/ncomms8965

    Figure Lengend Snippet: ( a ) Western blot analysis of SFK activation in HUVEC in response to TNF-α stimulation (10 ng ml −1 ). A phospho-specific antibody against Y416 in SFKs (pY416SFK) was used as an indicator of SFK activation. SFK activation was blocked with the pharmacological antagonist PP2 (10 μM) but not by its inactive analogue PP3 (10 μM). Antibody specificity for the phosphorylated form of the kinases was confirmed by dephosphorylating proteins in cell lysates with alkaline phosphatase. * P <0.05 as compared with unstimulated control (lane 1) and # P <0.001 as compared with lane 1 ( n =3). ( b ) TNF-α-induced ATP release from HUVEC following SFK inhibition with PP2. * P <0.05 versus control and PP3 treatments ( n =5). ( c ) Topological schematic of Panx1 highlighting an epitope in the intracellular loop which contains tyrosine 198. This epitope was used to develop antibodies specific to the phosphorylated (pY198Panx1) and non-phosphorylated (Panx1-IL) forms of the protein. ( d ) Overlay of pY198Panx1 signal and Panx1-IL (total) signal as assessed by western blotting with LiCOR IRDye secondary antibodies. pY198Panx1 detects a single species at ∼55 kDa. ( e ) Western blot analysis of pY198Panx1 in HUVEC transfected with plasmids encoding c-Src and/or inhibitor of Src (i-Src). ( f ) Western blot analysis of Panx1 phosphorylation at Y198 in HUVECs stimulated with TNF-α (10 ng ml −1 ). Phospho-signal was normalized to total Panx1 expression using the Panx1-IL Ab. * P <0.05 compared with vehicle control (lane 1) and # P <0.01 compared with 5-min TNF-α stimulation (lane 4) ( n =3). ( g ) TNF-α-induced ATP release from mesenteric venules treated with PP2 (10 μM) or PP3 (10 μM). * P <0.05 as compared with vehicle control ( n =5). ( h , i ) Western blot analysis of TNF-α-induced SFK activation ( h ) and pY198Panx1 phosphorylation ( i ) in isolated mesenteric venules perfused with TNF-α (50 ng ml −1 ) for 30 min. ( j ) Immunofluorescence micrographs of pY198Panx1 in isolated mesenteric venule cross-sections. Venules were isolated from mice expressing endogenous Panx1 in the vascular wall ( VECadER T2+ /Panx1 fl/fl +peanut oil) or mice with specific EC Panx1 deletion ( VECadER T2+ /Panx1 fl/fl +tamoxifen) and stimulated with TNF-α. Asterisks indicate the vessel lumen and nuclei are stained with 4,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 30 μm. All data are presented as mean±s.e.m. (error bars). Statistical analyses were performed using one-way analysis of variance.

    Article Snippet: To assess the role of SFK, cells were incubated with the SFK inhibitor PP2 (10 μM; Tocris) or its inactive analogue PP3 (10 μM; Tocris).

    Techniques: Western Blot, Activation Assay, Inhibition, Transfection, Expressing, Isolation, Immunofluorescence, Staining