staurosporine  (Alomone Labs)


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    Structured Review

    Alomone Labs staurosporine
    Application of protein kinase inhibitor <t>staurosporine</t> only inhibits the endocytosis induced by SQP25ms and SQP50ms: (A), Representative examples of Cm recordings obtained in response to the application of APls (i) and SQP50ms (ii) in two independent cells bathed in standard external solution (with addition of DMSO) (Control, black) or in presence of 100 nM staurosporine (red). (B), (C) and (D), The plots represent average values, standard errors and individual measurements (one measurement/cell) of ΔCm endo , endo/exo ratio and τ endo obtained by application of APls, SQP5ms, SQP10ms, SQP25ms and SQP50ms in Control (black) and staurosporine (red). The data were analyzed by Student’s ‘t’ test. *p
    Staurosporine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/staurosporine/product/Alomone Labs
    Average 93 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    staurosporine - by Bioz Stars, 2022-12
    93/100 stars

    Images

    1) Product Images from "Membrane Retrieval after Immediately Releasable Pool (IRP) Exocytosis is produced by Dynamin-Dependent and Dynamin-Independent Mechanisms"

    Article Title: Membrane Retrieval after Immediately Releasable Pool (IRP) Exocytosis is produced by Dynamin-Dependent and Dynamin-Independent Mechanisms

    Journal: bioRxiv

    doi: 10.1101/2022.08.31.506099

    Application of protein kinase inhibitor staurosporine only inhibits the endocytosis induced by SQP25ms and SQP50ms: (A), Representative examples of Cm recordings obtained in response to the application of APls (i) and SQP50ms (ii) in two independent cells bathed in standard external solution (with addition of DMSO) (Control, black) or in presence of 100 nM staurosporine (red). (B), (C) and (D), The plots represent average values, standard errors and individual measurements (one measurement/cell) of ΔCm endo , endo/exo ratio and τ endo obtained by application of APls, SQP5ms, SQP10ms, SQP25ms and SQP50ms in Control (black) and staurosporine (red). The data were analyzed by Student’s ‘t’ test. *p
    Figure Legend Snippet: Application of protein kinase inhibitor staurosporine only inhibits the endocytosis induced by SQP25ms and SQP50ms: (A), Representative examples of Cm recordings obtained in response to the application of APls (i) and SQP50ms (ii) in two independent cells bathed in standard external solution (with addition of DMSO) (Control, black) or in presence of 100 nM staurosporine (red). (B), (C) and (D), The plots represent average values, standard errors and individual measurements (one measurement/cell) of ΔCm endo , endo/exo ratio and τ endo obtained by application of APls, SQP5ms, SQP10ms, SQP25ms and SQP50ms in Control (black) and staurosporine (red). The data were analyzed by Student’s ‘t’ test. *p

    Techniques Used:

    Protein kinase inhibitor staurosporine abolishes the slow component of IRP replenishment. In order to determine the kinetics of IRP replenishment, a pair of 50 ms depolarizations (SQP50ms 1 and SQP50ms 2 , from −80 to +10 mV), with a variable time interval (Δt) between them, was applied in control conditions (A) and in presence of 100 nM staurosporine (B). The scheme of the stimulation protocol is represented at the top left of panel A. (i), I Ca2+ (above) and Cm (below) recordings obtained during a typical experiment when Δt = 5 s was applied. (ii), Relative replenishment of IRP (expressed as averages of ΔCm 2 /ΔCm 1 for every Δt applied) was plotted against the Δt between paired SQP50ms pulses and fitted to a biexponential growing function of the form (A) or (B). The fitting parameters for the control condition (A) were Y 0 =0.32±0.01, A 1 =0.35±0.03, τ 1 =1.14±0.13 s, A 2 =0.33±0.03, and τ 2 =8.57±1.14 s, R 2 > 0.9993; and for staurosporine (B) were Y 0 =0.25±0.03, A=0.73±0.03, and τ=0.72±0.06 s, R 2 > 0.9827. The points represented in the plots are averages of all the measurements obtained in individual cells (one measurement per cell). The number of individual cells measured (between parentheses) were: for (A), 0.2 s (7), 0.4 s (7), 1 s (6), 2 s (6), 3 s (6), 5 s (7), 7 s (8), 10 s (7), 20 s (7) and 40 s (7); and for (B), 0.2 s (11), 0.4 s (11), 1 s (12), 2 s (12), 3 s (11), 5 s (11), 7 s (12), 10 s (10), 20 s (12) and 40 s (9), obtained in 9 independent cell culture preparations. The fittings were performed on average values.
    Figure Legend Snippet: Protein kinase inhibitor staurosporine abolishes the slow component of IRP replenishment. In order to determine the kinetics of IRP replenishment, a pair of 50 ms depolarizations (SQP50ms 1 and SQP50ms 2 , from −80 to +10 mV), with a variable time interval (Δt) between them, was applied in control conditions (A) and in presence of 100 nM staurosporine (B). The scheme of the stimulation protocol is represented at the top left of panel A. (i), I Ca2+ (above) and Cm (below) recordings obtained during a typical experiment when Δt = 5 s was applied. (ii), Relative replenishment of IRP (expressed as averages of ΔCm 2 /ΔCm 1 for every Δt applied) was plotted against the Δt between paired SQP50ms pulses and fitted to a biexponential growing function of the form (A) or (B). The fitting parameters for the control condition (A) were Y 0 =0.32±0.01, A 1 =0.35±0.03, τ 1 =1.14±0.13 s, A 2 =0.33±0.03, and τ 2 =8.57±1.14 s, R 2 > 0.9993; and for staurosporine (B) were Y 0 =0.25±0.03, A=0.73±0.03, and τ=0.72±0.06 s, R 2 > 0.9827. The points represented in the plots are averages of all the measurements obtained in individual cells (one measurement per cell). The number of individual cells measured (between parentheses) were: for (A), 0.2 s (7), 0.4 s (7), 1 s (6), 2 s (6), 3 s (6), 5 s (7), 7 s (8), 10 s (7), 20 s (7) and 40 s (7); and for (B), 0.2 s (11), 0.4 s (11), 1 s (12), 2 s (12), 3 s (11), 5 s (11), 7 s (12), 10 s (10), 20 s (12) and 40 s (9), obtained in 9 independent cell culture preparations. The fittings were performed on average values.

    Techniques Used: Cell Culture

    2) Product Images from "Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity"

    Article Title: Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA120.014288

    The nonspecific kinase inhibitor staurosporine reduces the hyperpolarized shift of the I848T mutation . A , voltage dependence of activation using 500 nM staurosporine, incubated for 20 min. Staurosporine reduces the hyperpolarized shift of the I848T mutation from −9.77 ± 1.45 to −6.67 ± 1.54 mV. B , V 1/2 of channel activation (ANOVA F = 25.67; WT versus I848T p
    Figure Legend Snippet: The nonspecific kinase inhibitor staurosporine reduces the hyperpolarized shift of the I848T mutation . A , voltage dependence of activation using 500 nM staurosporine, incubated for 20 min. Staurosporine reduces the hyperpolarized shift of the I848T mutation from −9.77 ± 1.45 to −6.67 ± 1.54 mV. B , V 1/2 of channel activation (ANOVA F = 25.67; WT versus I848T p

    Techniques Used: Mutagenesis, Activation Assay, Incubation

    3) Product Images from "Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis"

    Article Title: Cdk5 and GSK3β inhibit Fast Endophilin-Mediated Endocytosis

    Journal: bioRxiv

    doi: 10.1101/2020.04.11.036863

    Acute inhibition of Cdk5 and GSK3 activates FEME. a , Scoring criteria used in the kinase screen. Representative images of ‘decreased’, ‘normal’ and ‘increased’ FEME in resting human RPE1 cells treated with 10μM dobutamine, 10μM DMSO and 10 nM GDC-0941 (PI3Ki), respectively. Arrowheads point at FEME carriers. ‘Decreased’ FEME was assigned for samples with > 80% reduction in the number of EPAs, in at least 50% of the cells. ‘Increased’ FEME was attributed to samples with > 200% elevation in the number of EPAs, in at least 50% of the cells. The corresponding scoring marks were 0, 1 and 2, respectively. b , Kinase screen using small compound inhibitors. RPE1 cells grown in complete medium were incubated for 10min at 37°C with the following inhibitors: DMSO, (vehicle); dobutamine, 10μM (positive control); Dinaciclib (Cdk1/2/5/9i), 1μM; CHIR-99041 (GSK3i1), 1μM; BIO (GSK3i2), 1μM; Roscovitine (Cdk1/2/5i), 1 m M; PHA-793887 (Cdk2/5/7i), 100nM; VX-745 (p38i), 10μM; JNK-IN-8 (JNKi), 1μM; staurosporine (broad kinases), 1μM; GNE-7915 (LRRK2i), 1μM; GSK2334470 (PDKi), 10μM; PF-4708671 (p70S6Ki), 10μM; AZ191 (DYRKi), 10μM; AZD0530 (SRCi), 1μM; TAK-632 (panRAFi), 10μM; GW 5074 (CRAFi), 1μM; PD0332991 (Cdk4/6i), 1μM; MK2206 (AKTi), 1μM; GDC-0879 (BRAFi), 1μM; CX-4945 (CK2i), 1μM; ZM 447439 (AurA/AurBi), 1μM; RO-3306 (Cdk1i), 100nM; BI 2536 (PLKi), 1μM; PD0325901 (MEKi), 100nM; Genistein (Y-kinases), 1μM; Purvalanol A (Cdk1/2/4i), 100nM; MLR 1023 (LYNi), 1μM; CDK1/2 inhibitor III (Cdk1/2i), 100nM; KT 5720 (PKAi), 100nM; BI-D1870 (p90RSKi), 100nM; PF-4800567 (CK1Ei), 1μM; SCH772984 (ERKi), 100nM; STO609 (CaMKK1/2ii), 100nM; P505-15 (SYKi), 1μM; PND-1186 (FAKi), 100nM; Torin 1 (mTORC1/2i), 10μM and GDC-0941 (PI3Ki), 100nM (negative control). c , Number of FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) upon titration of CHIR-99021, BIO, Roscovitine and Dinaciclib. Dobutamine and GDC-0941 were used as positive and negative controls, respectively. d , β1-adrenergic receptor (β1AR) uptake into FEME carriers in RPE1 cells pre-treated with 5μM CHIR-99021 (GSK3i) for 5 min, followed by 10μM dobutamine for 4 min or not (resting). Histograms show the mean ± SEM of the number of FEME carriers (left axis) and the number of FEME carriers positive for β1AR per 100 μm 2 (right axis) ( n =30 cells per condition, from biological triplicates). Arrowheads point at FEME carriers. All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA (b, and c) or two-way ANOVA (d); NS , non significant; *, P
    Figure Legend Snippet: Acute inhibition of Cdk5 and GSK3 activates FEME. a , Scoring criteria used in the kinase screen. Representative images of ‘decreased’, ‘normal’ and ‘increased’ FEME in resting human RPE1 cells treated with 10μM dobutamine, 10μM DMSO and 10 nM GDC-0941 (PI3Ki), respectively. Arrowheads point at FEME carriers. ‘Decreased’ FEME was assigned for samples with > 80% reduction in the number of EPAs, in at least 50% of the cells. ‘Increased’ FEME was attributed to samples with > 200% elevation in the number of EPAs, in at least 50% of the cells. The corresponding scoring marks were 0, 1 and 2, respectively. b , Kinase screen using small compound inhibitors. RPE1 cells grown in complete medium were incubated for 10min at 37°C with the following inhibitors: DMSO, (vehicle); dobutamine, 10μM (positive control); Dinaciclib (Cdk1/2/5/9i), 1μM; CHIR-99041 (GSK3i1), 1μM; BIO (GSK3i2), 1μM; Roscovitine (Cdk1/2/5i), 1 m M; PHA-793887 (Cdk2/5/7i), 100nM; VX-745 (p38i), 10μM; JNK-IN-8 (JNKi), 1μM; staurosporine (broad kinases), 1μM; GNE-7915 (LRRK2i), 1μM; GSK2334470 (PDKi), 10μM; PF-4708671 (p70S6Ki), 10μM; AZ191 (DYRKi), 10μM; AZD0530 (SRCi), 1μM; TAK-632 (panRAFi), 10μM; GW 5074 (CRAFi), 1μM; PD0332991 (Cdk4/6i), 1μM; MK2206 (AKTi), 1μM; GDC-0879 (BRAFi), 1μM; CX-4945 (CK2i), 1μM; ZM 447439 (AurA/AurBi), 1μM; RO-3306 (Cdk1i), 100nM; BI 2536 (PLKi), 1μM; PD0325901 (MEKi), 100nM; Genistein (Y-kinases), 1μM; Purvalanol A (Cdk1/2/4i), 100nM; MLR 1023 (LYNi), 1μM; CDK1/2 inhibitor III (Cdk1/2i), 100nM; KT 5720 (PKAi), 100nM; BI-D1870 (p90RSKi), 100nM; PF-4800567 (CK1Ei), 1μM; SCH772984 (ERKi), 100nM; STO609 (CaMKK1/2ii), 100nM; P505-15 (SYKi), 1μM; PND-1186 (FAKi), 100nM; Torin 1 (mTORC1/2i), 10μM and GDC-0941 (PI3Ki), 100nM (negative control). c , Number of FEME carriers (cytoplasmic Endophilin-positive assemblies, EPAs) upon titration of CHIR-99021, BIO, Roscovitine and Dinaciclib. Dobutamine and GDC-0941 were used as positive and negative controls, respectively. d , β1-adrenergic receptor (β1AR) uptake into FEME carriers in RPE1 cells pre-treated with 5μM CHIR-99021 (GSK3i) for 5 min, followed by 10μM dobutamine for 4 min or not (resting). Histograms show the mean ± SEM of the number of FEME carriers (left axis) and the number of FEME carriers positive for β1AR per 100 μm 2 (right axis) ( n =30 cells per condition, from biological triplicates). Arrowheads point at FEME carriers. All experiments were repeated at least three times with similar results. Statistical analysis was performed by one-way ANOVA (b, and c) or two-way ANOVA (d); NS , non significant; *, P

    Techniques Used: Inhibition, Incubation, Positive Control, Negative Control, Titration

    4) Product Images from "Sulfated Glycosphingolipid as Mediator of Phagocytosis: SM4s Enhances Apoptotic Cell Clearance and Modulates Macrophage Activity"

    Article Title: Sulfated Glycosphingolipid as Mediator of Phagocytosis: SM4s Enhances Apoptotic Cell Clearance and Modulates Macrophage Activity

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi:

    Comparative analysis of uptake of apoptotic cells from different cell lines, distinct apoptotic inducers, and different apoptotic stages. Late apoptotic C26 cells induced by cycloheximide ( left ), late apoptotic Renca cells induced by staurosporine ( middle ), and early apoptotic C26 cells ( right ) were subjected to flow cytometric analysis for PS exposure on the cell surface and membrane permeability ( upper lane ) and cell size and granularity ( middle lane ). The cells were used in FACS phagocytosis assay as described ( bottom lane ). Data in the graphs are presented as mean ± SEM of three experiments in duplicates. Statistical differences were evaluated by paired t test. *, p
    Figure Legend Snippet: Comparative analysis of uptake of apoptotic cells from different cell lines, distinct apoptotic inducers, and different apoptotic stages. Late apoptotic C26 cells induced by cycloheximide ( left ), late apoptotic Renca cells induced by staurosporine ( middle ), and early apoptotic C26 cells ( right ) were subjected to flow cytometric analysis for PS exposure on the cell surface and membrane permeability ( upper lane ) and cell size and granularity ( middle lane ). The cells were used in FACS phagocytosis assay as described ( bottom lane ). Data in the graphs are presented as mean ± SEM of three experiments in duplicates. Statistical differences were evaluated by paired t test. *, p

    Techniques Used: Flow Cytometry, Permeability, FACS, Phagocytosis Assay

    C26 and Renca cells demonstrate late apoptotic phenotype after induction with staurosporine and successful SM4s painting after exposure to SM4s. A , C26 cells were incubated with staurosporine (1 µ M) for 24 h. PS exposure and membrane permeability were detected by flow cytometry using FITC-conjugated annexin V and PI, respectively. Annexin V/PI double-positive cells were considered as late apoptotic. B , Electron photomicrograph of apoptotic C26 cell with evident nuclear condensation and fragmentation (Zeiss, ×30000). B and C , Electron photomicrographs of apoptotic C26 cells preincubated with 10 µ M SM4s. Gold particle-conjugated goat anti-mouse Ab (particle size of 6 nm) was used for visualization of the sulfatide (arrows). D–F , Apoptotic C26 cells were prepared as described above, spun down, stained with DRAQ5 (nucleus), and immunostained with anti-O4 and AlexaFluor 488 donkey anti-mouse IgG (H+L) mAbs (SM4s). F , Confocal image of a single cell shows predominant membrane localization of the sulfatide (×5000). Data are presented as merge of green and blue channels. G–L , Apoptotic cells subjected to immunocytochemistry analysis with anti-O4 mAb. Photomicrographs of apoptotic cells treated with sulfatide or vehicle (DMSO). G and J , C26 and Renca control cells (Leica, ×400). H and I , C26 cells treated with 10 µ M SM4s (Leica, ×400 and ×1000, respectively). K , Late apoptotic Renca cells painted with sulfatide (Leica, ×400). L , Binding of SM4s to plasma membrane of late apoptotic (arrows) and early apoptotic (star) Renca cells (Leica, ×400).
    Figure Legend Snippet: C26 and Renca cells demonstrate late apoptotic phenotype after induction with staurosporine and successful SM4s painting after exposure to SM4s. A , C26 cells were incubated with staurosporine (1 µ M) for 24 h. PS exposure and membrane permeability were detected by flow cytometry using FITC-conjugated annexin V and PI, respectively. Annexin V/PI double-positive cells were considered as late apoptotic. B , Electron photomicrograph of apoptotic C26 cell with evident nuclear condensation and fragmentation (Zeiss, ×30000). B and C , Electron photomicrographs of apoptotic C26 cells preincubated with 10 µ M SM4s. Gold particle-conjugated goat anti-mouse Ab (particle size of 6 nm) was used for visualization of the sulfatide (arrows). D–F , Apoptotic C26 cells were prepared as described above, spun down, stained with DRAQ5 (nucleus), and immunostained with anti-O4 and AlexaFluor 488 donkey anti-mouse IgG (H+L) mAbs (SM4s). F , Confocal image of a single cell shows predominant membrane localization of the sulfatide (×5000). Data are presented as merge of green and blue channels. G–L , Apoptotic cells subjected to immunocytochemistry analysis with anti-O4 mAb. Photomicrographs of apoptotic cells treated with sulfatide or vehicle (DMSO). G and J , C26 and Renca control cells (Leica, ×400). H and I , C26 cells treated with 10 µ M SM4s (Leica, ×400 and ×1000, respectively). K , Late apoptotic Renca cells painted with sulfatide (Leica, ×400). L , Binding of SM4s to plasma membrane of late apoptotic (arrows) and early apoptotic (star) Renca cells (Leica, ×400).

    Techniques Used: Incubation, Permeability, Flow Cytometry, Cytometry, Staining, Immunocytochemistry, Binding Assay

    5) Product Images from "Protein kinase C enhances the rapidly activating delayed rectifier potassium current, IKr, through a reduction in C-type inactivation in guinea-pig ventricular myocytes"

    Article Title: Protein kinase C enhances the rapidly activating delayed rectifier potassium current, IKr, through a reduction in C-type inactivation in guinea-pig ventricular myocytes

    Journal: The Journal of Physiology

    doi: 10.1111/j.1469-7793.2000.t01-2-00391.x

    The role of protein kinases in the forskolin- and isoprenaline-induced increase in I Kr Aa , effect of 3 μM staurosporine on I Kr tail current amplitude and the effect of forskolin (5 μM) in the continued presence of staurosporine ( n = 4). The protein kinase C inhibitor bisindolylmaleimide I (100 nM) completely inhibited the increase in I Kr induced by forskolin ( Ab ) and isoprenaline ( Ac) . Ad , phorbol dibutyrate (100 nM) increased I Kr tail current amplitude. B , the effect of isoprenaline on I Kr (upper traces) and I Ca (lower traces) in the presence of bisindolylmaleimide I (100 nM). I Kr and I Ca in B were recorded from the same cells and the arrows indicate 100 pA above zero current in the upper figures and zero current in the lower figures. * P
    Figure Legend Snippet: The role of protein kinases in the forskolin- and isoprenaline-induced increase in I Kr Aa , effect of 3 μM staurosporine on I Kr tail current amplitude and the effect of forskolin (5 μM) in the continued presence of staurosporine ( n = 4). The protein kinase C inhibitor bisindolylmaleimide I (100 nM) completely inhibited the increase in I Kr induced by forskolin ( Ab ) and isoprenaline ( Ac) . Ad , phorbol dibutyrate (100 nM) increased I Kr tail current amplitude. B , the effect of isoprenaline on I Kr (upper traces) and I Ca (lower traces) in the presence of bisindolylmaleimide I (100 nM). I Kr and I Ca in B were recorded from the same cells and the arrows indicate 100 pA above zero current in the upper figures and zero current in the lower figures. * P

    Techniques Used:

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    Alomone Labs staurosporine
    Application of protein kinase inhibitor <t>staurosporine</t> only inhibits the endocytosis induced by SQP25ms and SQP50ms: (A), Representative examples of Cm recordings obtained in response to the application of APls (i) and SQP50ms (ii) in two independent cells bathed in standard external solution (with addition of DMSO) (Control, black) or in presence of 100 nM staurosporine (red). (B), (C) and (D), The plots represent average values, standard errors and individual measurements (one measurement/cell) of ΔCm endo , endo/exo ratio and τ endo obtained by application of APls, SQP5ms, SQP10ms, SQP25ms and SQP50ms in Control (black) and staurosporine (red). The data were analyzed by Student’s ‘t’ test. *p
    Staurosporine, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/staurosporine/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    staurosporine - by Bioz Stars, 2022-12
    93/100 stars
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    Application of protein kinase inhibitor staurosporine only inhibits the endocytosis induced by SQP25ms and SQP50ms: (A), Representative examples of Cm recordings obtained in response to the application of APls (i) and SQP50ms (ii) in two independent cells bathed in standard external solution (with addition of DMSO) (Control, black) or in presence of 100 nM staurosporine (red). (B), (C) and (D), The plots represent average values, standard errors and individual measurements (one measurement/cell) of ΔCm endo , endo/exo ratio and τ endo obtained by application of APls, SQP5ms, SQP10ms, SQP25ms and SQP50ms in Control (black) and staurosporine (red). The data were analyzed by Student’s ‘t’ test. *p

    Journal: bioRxiv

    Article Title: Membrane Retrieval after Immediately Releasable Pool (IRP) Exocytosis is produced by Dynamin-Dependent and Dynamin-Independent Mechanisms

    doi: 10.1101/2022.08.31.506099

    Figure Lengend Snippet: Application of protein kinase inhibitor staurosporine only inhibits the endocytosis induced by SQP25ms and SQP50ms: (A), Representative examples of Cm recordings obtained in response to the application of APls (i) and SQP50ms (ii) in two independent cells bathed in standard external solution (with addition of DMSO) (Control, black) or in presence of 100 nM staurosporine (red). (B), (C) and (D), The plots represent average values, standard errors and individual measurements (one measurement/cell) of ΔCm endo , endo/exo ratio and τ endo obtained by application of APls, SQP5ms, SQP10ms, SQP25ms and SQP50ms in Control (black) and staurosporine (red). The data were analyzed by Student’s ‘t’ test. *p

    Article Snippet: MaterialsBovine serum albumin (CAT No. 05470-25G), poly-L-lysine (CAT No. P8920-100ML), cytosine-1-beta-D-arabinofuranoside (CAT No. C6645-25MG), papain (CAT No. P4762-100MG), Mg ATP (CAT No. A9187-100MG), Li GTP (CAT No. G5884-25MG), EGTA (CAT No. E4378-10G), GTPγS (G8634-1MG), bisindolimaleimide XI (BIS XI) (B4056-1MG) and phorbol 12-myristate 13-acetate (PMA) (P8139-1MG) were obtained from Sigma (St Louis, MO, USA); Dulbecco’s modified Eagle’s medium (CAT No. 10567-014), gentamycin (CAT No. 15750078) and penicillin/streptomycin (CAT No. 15140122) from Gibco (Carlsbad, CA, USA); fetal calf serum from Natocor (Córdoba, Argentina, CAT No. SFB500ml); tetrodotoxin citrate (CAT No. T-550) and staurosporine (S-350) from Alomone Labs (Jerusalem, Israel); and the monoclonal anti-dynamin antibody (BD Biosciences, San Jose, CA, EEUU, CAT No. 610246, RRID:AB_397641).

    Techniques:

    Protein kinase inhibitor staurosporine abolishes the slow component of IRP replenishment. In order to determine the kinetics of IRP replenishment, a pair of 50 ms depolarizations (SQP50ms 1 and SQP50ms 2 , from −80 to +10 mV), with a variable time interval (Δt) between them, was applied in control conditions (A) and in presence of 100 nM staurosporine (B). The scheme of the stimulation protocol is represented at the top left of panel A. (i), I Ca2+ (above) and Cm (below) recordings obtained during a typical experiment when Δt = 5 s was applied. (ii), Relative replenishment of IRP (expressed as averages of ΔCm 2 /ΔCm 1 for every Δt applied) was plotted against the Δt between paired SQP50ms pulses and fitted to a biexponential growing function of the form (A) or (B). The fitting parameters for the control condition (A) were Y 0 =0.32±0.01, A 1 =0.35±0.03, τ 1 =1.14±0.13 s, A 2 =0.33±0.03, and τ 2 =8.57±1.14 s, R 2 > 0.9993; and for staurosporine (B) were Y 0 =0.25±0.03, A=0.73±0.03, and τ=0.72±0.06 s, R 2 > 0.9827. The points represented in the plots are averages of all the measurements obtained in individual cells (one measurement per cell). The number of individual cells measured (between parentheses) were: for (A), 0.2 s (7), 0.4 s (7), 1 s (6), 2 s (6), 3 s (6), 5 s (7), 7 s (8), 10 s (7), 20 s (7) and 40 s (7); and for (B), 0.2 s (11), 0.4 s (11), 1 s (12), 2 s (12), 3 s (11), 5 s (11), 7 s (12), 10 s (10), 20 s (12) and 40 s (9), obtained in 9 independent cell culture preparations. The fittings were performed on average values.

    Journal: bioRxiv

    Article Title: Membrane Retrieval after Immediately Releasable Pool (IRP) Exocytosis is produced by Dynamin-Dependent and Dynamin-Independent Mechanisms

    doi: 10.1101/2022.08.31.506099

    Figure Lengend Snippet: Protein kinase inhibitor staurosporine abolishes the slow component of IRP replenishment. In order to determine the kinetics of IRP replenishment, a pair of 50 ms depolarizations (SQP50ms 1 and SQP50ms 2 , from −80 to +10 mV), with a variable time interval (Δt) between them, was applied in control conditions (A) and in presence of 100 nM staurosporine (B). The scheme of the stimulation protocol is represented at the top left of panel A. (i), I Ca2+ (above) and Cm (below) recordings obtained during a typical experiment when Δt = 5 s was applied. (ii), Relative replenishment of IRP (expressed as averages of ΔCm 2 /ΔCm 1 for every Δt applied) was plotted against the Δt between paired SQP50ms pulses and fitted to a biexponential growing function of the form (A) or (B). The fitting parameters for the control condition (A) were Y 0 =0.32±0.01, A 1 =0.35±0.03, τ 1 =1.14±0.13 s, A 2 =0.33±0.03, and τ 2 =8.57±1.14 s, R 2 > 0.9993; and for staurosporine (B) were Y 0 =0.25±0.03, A=0.73±0.03, and τ=0.72±0.06 s, R 2 > 0.9827. The points represented in the plots are averages of all the measurements obtained in individual cells (one measurement per cell). The number of individual cells measured (between parentheses) were: for (A), 0.2 s (7), 0.4 s (7), 1 s (6), 2 s (6), 3 s (6), 5 s (7), 7 s (8), 10 s (7), 20 s (7) and 40 s (7); and for (B), 0.2 s (11), 0.4 s (11), 1 s (12), 2 s (12), 3 s (11), 5 s (11), 7 s (12), 10 s (10), 20 s (12) and 40 s (9), obtained in 9 independent cell culture preparations. The fittings were performed on average values.

    Article Snippet: MaterialsBovine serum albumin (CAT No. 05470-25G), poly-L-lysine (CAT No. P8920-100ML), cytosine-1-beta-D-arabinofuranoside (CAT No. C6645-25MG), papain (CAT No. P4762-100MG), Mg ATP (CAT No. A9187-100MG), Li GTP (CAT No. G5884-25MG), EGTA (CAT No. E4378-10G), GTPγS (G8634-1MG), bisindolimaleimide XI (BIS XI) (B4056-1MG) and phorbol 12-myristate 13-acetate (PMA) (P8139-1MG) were obtained from Sigma (St Louis, MO, USA); Dulbecco’s modified Eagle’s medium (CAT No. 10567-014), gentamycin (CAT No. 15750078) and penicillin/streptomycin (CAT No. 15140122) from Gibco (Carlsbad, CA, USA); fetal calf serum from Natocor (Córdoba, Argentina, CAT No. SFB500ml); tetrodotoxin citrate (CAT No. T-550) and staurosporine (S-350) from Alomone Labs (Jerusalem, Israel); and the monoclonal anti-dynamin antibody (BD Biosciences, San Jose, CA, EEUU, CAT No. 610246, RRID:AB_397641).

    Techniques: Cell Culture

    Acute inhibition of Cdk5 and GSK3 activates FEME. a Scoring criteria used in the kinase screen. Representative images of decreased, normal, and increased FEME in resting human RPE1 cells treated with 10 μM dobutamine, 10 μM DMSO, and 10 nM GDC-0941 (PI3Ki), respectively. Arrowheads point at FEME carriers. Decreased FEME was assigned for samples with > 80% reduction in the number of cytoplasmic Endophilin-positive assemblies (EPAs), in at least 50% of the cells. Increased FEME was attributed to samples with > 200% elevation in the number of EPAs, in at least 50% of the cells. The corresponding scoring marks were 0, 1, and 2, respectively. Scale bar, 5 μm. b Kinase screen using small compound inhibitors. RPE1 cells grown in complete medium were incubated for 10 min at 37 °C with the following inhibitors: DMSO, (vehicle); dobutamine, 10 μM (positive control); Dinaciclib (Cdk1/2/5/9i), 1 μM; CHIR-99041 (GSK3i1), 1 μM; BIO (GSK3i2), 1μM; Roscovitine (Cdk1/2/5i), 1 μM; PHA-793887 (Cdk2/5/7i), 100 nM; VX-745 (p38i), 10 μM; JNK-IN-8 (JNKi), 1 μM; staurosporine (broad kinases), 1 μM; GNE-7915 (LRRK2i), 1 μM; AZ191 (DYRK1Bi), 10 μM; GSK2334470 (PDKi), 10 μM; PF-4708671 (p70S6Ki), 10μM; AZ191 (DYRKi), 10 μM; AZD0530 (broad SRCi), 1 μM; TAK-632 (panRAFi), 10 μM; GW 5074 (CRAFi), 1 μM; PD0332991 (Cdk4/6i), 1 μM; MK2206 (AKTi), 1 μM; GDC-0879 (BRAFi), 1 μM; CX-4945 (CK2i), 1 μM; ZM 447439 (AurA/AurBi), 1 μM; RO-3306 (Cdk1i), 100 nM; BI 2536 (PLKi), 1 μM; PD0325901 (MEKi), 100 nM; Genistein (Y-kinases), 1 μM; Purvalanol A (Cdk1/2/4i), 100 nM; MLR 1023 (LYNi), 1 μM; P505-15 (SYKi), 100 nM; CDK1/2 inhibitor III (Cdk1/2i), 100 nM; KT 5720 (PKAi), 100 nM; BI-D1870 (p90RSKi), 100 nM; D4476 (CK1E), 1 μM; PF-4800567 (CK1Ei), 1 μM; SCH772984 (ERKi), 100 nM; STO609 (CaMKK1/2ii), 100 nM; P505-15 (SYKi), 1μM; PND-1186 (FAKi), 100 nM; Torin 1 (mTORC1/2i), 10 μM and GDC-0941 (PI3Ki), 100 nM (negative control). Histograms show the mean ± SEM from 12 well per condition, from three independent biological experiments. Statistical analysis was performed by one-way ANOVA. ns non significant; * P

    Journal: Nature Communications

    Article Title: Cdk5 and GSK3β inhibit fast endophilin-mediated endocytosis

    doi: 10.1038/s41467-021-22603-4

    Figure Lengend Snippet: Acute inhibition of Cdk5 and GSK3 activates FEME. a Scoring criteria used in the kinase screen. Representative images of decreased, normal, and increased FEME in resting human RPE1 cells treated with 10 μM dobutamine, 10 μM DMSO, and 10 nM GDC-0941 (PI3Ki), respectively. Arrowheads point at FEME carriers. Decreased FEME was assigned for samples with > 80% reduction in the number of cytoplasmic Endophilin-positive assemblies (EPAs), in at least 50% of the cells. Increased FEME was attributed to samples with > 200% elevation in the number of EPAs, in at least 50% of the cells. The corresponding scoring marks were 0, 1, and 2, respectively. Scale bar, 5 μm. b Kinase screen using small compound inhibitors. RPE1 cells grown in complete medium were incubated for 10 min at 37 °C with the following inhibitors: DMSO, (vehicle); dobutamine, 10 μM (positive control); Dinaciclib (Cdk1/2/5/9i), 1 μM; CHIR-99041 (GSK3i1), 1 μM; BIO (GSK3i2), 1μM; Roscovitine (Cdk1/2/5i), 1 μM; PHA-793887 (Cdk2/5/7i), 100 nM; VX-745 (p38i), 10 μM; JNK-IN-8 (JNKi), 1 μM; staurosporine (broad kinases), 1 μM; GNE-7915 (LRRK2i), 1 μM; AZ191 (DYRK1Bi), 10 μM; GSK2334470 (PDKi), 10 μM; PF-4708671 (p70S6Ki), 10μM; AZ191 (DYRKi), 10 μM; AZD0530 (broad SRCi), 1 μM; TAK-632 (panRAFi), 10 μM; GW 5074 (CRAFi), 1 μM; PD0332991 (Cdk4/6i), 1 μM; MK2206 (AKTi), 1 μM; GDC-0879 (BRAFi), 1 μM; CX-4945 (CK2i), 1 μM; ZM 447439 (AurA/AurBi), 1 μM; RO-3306 (Cdk1i), 100 nM; BI 2536 (PLKi), 1 μM; PD0325901 (MEKi), 100 nM; Genistein (Y-kinases), 1 μM; Purvalanol A (Cdk1/2/4i), 100 nM; MLR 1023 (LYNi), 1 μM; P505-15 (SYKi), 100 nM; CDK1/2 inhibitor III (Cdk1/2i), 100 nM; KT 5720 (PKAi), 100 nM; BI-D1870 (p90RSKi), 100 nM; D4476 (CK1E), 1 μM; PF-4800567 (CK1Ei), 1 μM; SCH772984 (ERKi), 100 nM; STO609 (CaMKK1/2ii), 100 nM; P505-15 (SYKi), 1μM; PND-1186 (FAKi), 100 nM; Torin 1 (mTORC1/2i), 10 μM and GDC-0941 (PI3Ki), 100 nM (negative control). Histograms show the mean ± SEM from 12 well per condition, from three independent biological experiments. Statistical analysis was performed by one-way ANOVA. ns non significant; * P

    Article Snippet: Small compound inhibitors and ligands The following small compound inhibitors (amongst the best-reported inhibitors for each kinase , ) were used: AZ191 (called DYRKi in this study Cayman 17693), AZD0530 aka Sacratinib (called SRCi in this study, Cayman 11497), BI-D1870 (called p90RSKi in this study, Cayman 15264), BIO-6-bromoindirubin-3′-oxime, aka BIO (called GSK3i2 in this study, (Sigma B1686), BI 2536 (called PLKi in this study, Selleckchem S1109), CDK1/2 inhibitor III (called Cdk1/2i in this study, Merck 217714), CHIR-99041 (called GSK3i1 in this study, Cayman 13122), Ciliobrevin D (called Ciliobrevin in this study, Calbiochem 250401), CX-4945 (called CK2i in this study, Cayman 16779), Dinaciclib (called Cdk1/2/5/9i in this study, MedChemExpress Hy-10492), Dobutamine (Sigma D0676), D4476 (called CK1i in this study, BioVision 1770), GDC-0879 (called BRAFi in this study, Tocris 4453), GDC-0941 (called PI3Ki in this study, Symansis SYG0941), Genistein (called Y-kinases in this study, Calbiochem 245834), GNE-7915 (called LRRK2i in this study, MedChemExpress Hy-10328), GSK2334470 (called PDKi in this study, Cayman 18095), GW 5074 (called CRAFi in this study, Santa Crux sc-200639), Harmine hydrochloride (called DYRKi in this study, Santa Crux sc2595136), JNK-IN-8 (called JNKi in this study MedChemExpress Hy-13319), KT 5720 (called PKAi in this study Cayman 10011011), MK2206 (called AKTi in this study, LKT Laboratories M4000), MLR 1023 (called LYNa in this study, Tocris 4582), PD0325901 (called MEKi in this study, Tocris 4192), PD0332991 aka Palbociclib (called Cdk4/6i in this study, Sigma PZ0199), PF-4708671 (called p70S6Ki in this study, MedChemExpress Hy-15773), PF-4800567 (called CK1Ei in this study, Cayman 19171), PHA-793887 (called Cdk2/5/7i in this study, ApexBio A5459), PND-1186 (called FAKi in this study, MedChemExpress Hy-13917), Purvalanol A (called Cdk1/2/4i in this study, Santa Cruz sc-224244), P505-15 (called SYKi in this study, Adooq Bioscence A11952), Roscovitine (called Cdk1/2/5i in this study, Santa Cruz sc-24002), RO-3306 (called Cdk1i in this study, Cayman 15149), SCH772984 (called ERKi in this study, Sellekchem S7101), Staurosporine (called broad kinases in this study, Alomone Labs AM-2282), STO609 (called CaMKK1/2i in this study, Cayman 15325), TAK-632 (called panRAFi in this study, Selleckchem S7291), Torin 1 (called mTORC1i in this study, Tocris 4247), VX-745 (called p38i in this study, MedChemExpress Hy-10328) and ZM 447439 (called AurA/AurBi in this study, Cayman 13601).

    Techniques: Inhibition, Incubation, Positive Control, Negative Control

    The nonspecific kinase inhibitor staurosporine reduces the hyperpolarized shift of the I848T mutation . A , voltage dependence of activation using 500 nM staurosporine, incubated for 20 min. Staurosporine reduces the hyperpolarized shift of the I848T mutation from −9.77 ± 1.45 to −6.67 ± 1.54 mV. B , V 1/2 of channel activation (ANOVA F = 25.67; WT versus I848T p

    Journal: The Journal of Biological Chemistry

    Article Title: Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity

    doi: 10.1074/jbc.RA120.014288

    Figure Lengend Snippet: The nonspecific kinase inhibitor staurosporine reduces the hyperpolarized shift of the I848T mutation . A , voltage dependence of activation using 500 nM staurosporine, incubated for 20 min. Staurosporine reduces the hyperpolarized shift of the I848T mutation from −9.77 ± 1.45 to −6.67 ± 1.54 mV. B , V 1/2 of channel activation (ANOVA F = 25.67; WT versus I848T p

    Article Snippet: Staurosporine was purchased at Alomone labs (Cat.#: S-350) and stored protected from light.

    Techniques: Mutagenesis, Activation Assay, Incubation