inactive analogue pp3  (Millipore)

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

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

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

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

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