staurosporine (Alomone Labs)

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Alomone Labs staurosporine

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; <t>staurosporine</t> (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

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1) 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

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

2) 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:

3) 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

4) 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

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Journal: bioRxiv

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

doi: 10.1101/2020.04.11.036863

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 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

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), 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), 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 ‘CaMKK1/2ii’ 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, Titration

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

Journal: The Journal of Physiology

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

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

Figure Lengend 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

Article Snippet: The following were prepared as stock solutions and stored at either 4°C or −20°C as necessary: E4031 (Eisai, Tokyo, Japan) was dissolved in water, forskolin, phorbol dibutyrate, BAPTA-AM (Sigma), staurosporine (Alomone Labs, Israel), bisindolylmaleimide I and KT5720 (Calbiochem) in DMSO and nifedipine (Sigma) in ethanol.

Techniques:

Journal: Aging (Albany NY)

Article Title: BK channel overexpression on plasma membrane of fibroblasts from Hutchinson-Gilford progeria syndrome

doi: 10.18632/aging.101621

Figure Lengend Snippet: Immunofluorescence detection for BK Ca channels expressed on plasma membrane in living cells. ( A ) Fluorescence and fluorescence/phase contrast merged micrographs of isolated hDF obtained from young, elderly and HGPS donors incubated with an anti-BK Ca α subunit primary antibody visualized by the conjugated Alexa Fluor 350 fluorophore and acquired at 200× magnification. Scale bars: 100 μm. ( B ) Histogram showing the percentage of cells expressing a blue fluorescence intensity over a fixed threshold (% ± SEM). Significant differences calculated according to the Student’s t-test (p

Article Snippet: Living fibroblasts adhered in monolayer culture from Young, Elderly and HGPS groups were double labeled by using the custom synthesized rabbit polyclonal IgG specific to the extracellular amino acids residues KCNMA1-69:86 conjugated with Alexa Fluor 350 (Invitrogen, Life Technologies Europe, Milano, Italy), and the cell-permeant calcium (Ca2+) fluorescent probe Fluo-4 AM (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Techniques: Immunofluorescence, Fluorescence, Isolation, Incubation, Expressing