Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: Syk S297 phosphorylation is stoichiometric and completely blocked by PKC inhibition. Washed human platelets were pre-incubated with DMSO as vehicle control or with pan-PKC inhibitor GF109203X (GFX) (5 µM) at 37 °C for 5 min prior to stimulation with 50 ng/mL cvx. ( a ) Platelet aggregation was stopped by adding Laemmli buffer directly in the cuvettes after 1, 2, and 5 min of stimulation under stirring conditions, and Syk S297 phosphorylation was analyzed by standard SDS-PAGE/Western blot analysis compared to total Syk. Quantitative analyses are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, DMSO versus GFX-treated platelets at 1 min. ( bi–biii ) Washed human platelets were stimulated under non-stirring conditions at 37 °C, and platelets were lysed after the indicated time points by using Laemmli buffer for phos-tag SDS-PAGE. ( bi ) Samples were analyzed by phos-tag SDS-PAGE followed by immunoblotting using total Syk antibody. Same samples were analyzed by standard SDS-PAGE followed by immunoblotting using ( bii ) total Syk antibody or ( biii ) anti-Syk S297. Blots are representative of two independent experiments from two healthy donors.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Inhibition, Incubation, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: Syk S297 phosphorylation is upregulated by PKC activation in a stoichiometric manner. Washed human platelets were pre-incubated with GF109203X (5 µM) for 5 min at 37 °C prior to stimulation with the phorbol ester PDBu, a global PKC activator (0.2 µM). ( a ) Platelet aggregation was stopped by adding Laemmli buffer directly in the cuvettes after 15, 60, 120, and 300 s of stimulation under stirring conditions, and Syk S297 phosphorylation was analyzed by standard SDS-PAGE/Western blot analysis compared to total Syk. Quantitative analyses are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, untreated versus GFX treated platelets at 1 min. ( bi–biii ) Washed human platelets were stimulated under non-stirring conditions at 37 °C, and platelets were lysed after the indicated time points by using Laemmli buffer for phos-tag SDS-PAGE/immunoblotting analysis. ( bi ) Samples were analyzed by phos-tag SDS-PAGE followed by immunoblotting using total Syk antibody. Same samples were analyzed by standard SDS-PAGE followed by immunoblotting using ( bii ) total Syk antibody or ( biii ) anti-pSyk S297. Blots are representative of two independent experiments from two healthy donors.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Activation Assay, Incubation, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: PKC activation induces Syk S297 phosphorylation, which is independent from Syk and only partially inhibited by PKA. Washed human platelets were pre-incubated with DMSO (vehicle control) or with pan-PKC inhibitor, GF109203X (5 µM) at 37 °C for 5 min prior to stimulation with 0.2 µM PDBu or 50 ng/mL cvx. ( a ) Platelet aggregation was stopped by adding Laemmli buffer after 1 min of stimulation under stirring conditions. MARCKS S159/163 phosphorylation was analyzed by standard SDS-PAGE analysis compared to the loading control β-actin. Washed human platelets were pre-incubated with ( b ) Syk inhibitor, PRT-060318 (1 µM) for 5 min or ( c ) iloprost (2 nM) for 3 min prior to stimulation with PDBu. Syk S297 and MARCKS S159/163 phosphorylation was analyzed compared to total Syk or β-actin, respectively. The corresponding quantifications are represented as means ± S.D from three independent experiments with platelets from three healthy donors. **** p < 0.0001, ** p < 0.01, * p < 0.05, ns: not significant.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Activation Assay, Incubation, SDS Page

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: Syk phosphorylation is differentially regulated by PKC. Washed human platelets were pre-incubated with DMSO (vehicle control) or with pan-PKC inhibitor GF109203X (5 µM) at 37 °C for 5 min prior to stimulation with ( a ) 50 ng/mL cvx or ( b ) 0.5% ( v / v ) EB. Platelet aggregation was stopped by adding Laemmli buffer after 1, 2, or 5 min of stimulation under stirring conditions. The kinetics of the phosphorylation of Syk on ( ai , bi ) S297, ( aii , bii ) Y525/526, and ( aiii , biii ) Y352 compared to total Syk are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, DMSO-versus GFX-treated platelets at 1 min in response to cvx or EB.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Incubation

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: PKC inhibition induces hyperphosphorylation of Y-sites of Syk substrates. Washed human platelets were pre-incubated with DMSO (vehicle control) or with pan-PKC inhibitor GF109203X (5 µM) at 37 °C for 5 min prior to stimulation with ( a ) 50 ng/mL cvx or ( b ) 0.5% ( v / v ) EB. Platelet aggregation was stopped by adding Laemmli buffer after 1, 2, or 5 min of stimulation under stirring conditions. The kinetics of the phosphorylation of ( ai , bi ) LAT on Y191 and ( aii , bii ) PLCγ2 on Y759 compared to the adequate loading control are represented as means ± SD from three independent experiments with platelets from three healthy donors. # p < 0.0001, * p < 0.05, DMSO-versus GFX-treated platelets at 1 min or 2 min in response to cvx or EB, respectively.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Inhibition, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Feedback Regulation of Syk by Protein Kinase C in Human Platelets

doi: 10.3390/ijms21010176

Figure Lengend Snippet: PKC inhibition significantly enhances InsP1 accumulation and intracellular Ca 2+ mobilization. ( a ) Washed human platelets were pre-treated with GF109203X (5 µM) as previously described in the presence of 1 mM LiCl. Platelet aggregation was stopped after 5 min using lysis buffer provided by the manufacturer. ( b ) Washed human platelets were pre-incubated with Fluo-3 AM (5 µM) for 30 min at 37 °C, and platelet stimulation was performed directly before measurement. Ca 2+ i mobilization was monitored for 120 s by flow cytometry. Quantitative data are represented as means ± SD from three independent experiments with platelets from three healthy donors. **** p < 0.0001, * p < 0.05.

Article Snippet: Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 (Abcam, Cambridge, UK), Syk inhibitors OXSI-2 (Merck, Darmstadt, Germany) and PRT-060318 (Sellckem, Houston, TX, USA), PKC pan-inhibitor GF109203X (Enzo life sciences, Lausen, Switzerland), or cAMP-elevating agent iloprost (Bayer AG, Leverkusen, Germany).

Techniques: Inhibition, Lysis, Incubation, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: NHEJ repair was impaired by Cd ions through prolonged DNA-PKcs phosphorylation, and subsequently promoted dissociation of DNA-PKcs from DNA ends. ( A ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of Ku70 and Ku80 on the chromatin fraction. ( B ) U2OS cells were transfected with NHEJ reporter and/or I-SceI plasmids for 4 h, followed by treatment with or without CdCl 2 (100 μM) for 3 h, and ChIP assay was performed using anti-Ku80 antibody, followed by PCR to detect the Ku80 bound DSB fragment. ( C ) Immunoblotting of whole cell extracts from GC-2 cells treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) analyzed at the indicated times. ( D ) Immunoblotting of whole cell extracts from GC-2 cells treated by X-rays (2 Gy) with or without CdCl 2 (20 μM) analyzed at the indicated times. ( E ) U2OS cells were transfected with NHEJ reporter and/or I-SceI plasmids for 4 h, followed by treatment with or without CdCl 2 (100 μM) for 3 h, and ChIP assay was performed using anti-DNA-PKcs antibody, followed by PCR to detect DNA-PKcs bound DSB fragment. ( F ) U2OS cells were treated by H 2 O 2 (1 mM) with or without CdCl 2 (100 μM) for 1 h, followed by IP, with Ku80 antibody associated DNA-PKcs analyzed by western blot. ‘*’ indicates nonspecific band. ( G ) Quantitative analysis of DNA-PKcs binding with Ku80 by immunocoprecipitation in (F). ( H ) Immunoblotting of chromatin fraction from GC-2 cells treated by X-rays (2 Gy) with or without CdCl 2 (20 μM) analyzed at the indicated times. ( I ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of Artemis on the chromatin fraction. ** P < 0.01; *** P < 0.001; ns indicates non-significant difference. Data presented as mean ± SD from three independent experiments.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Phospho-proteomics, Western Blot, Transfection, Binding Assay

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: PP5 inactivation induced by Cd ions mediated the prolonged phosphorylation of DNA-PKcs. ( A ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of phosphorylation of ATM in whole cells. ( B ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, and/or pretreated with 200nM OA for 30 min, followed by immunoblot analysis of indicated protein in whole cells. ( C ) After siPP5 for 72 h, GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of indicated protein in whole cells. ( D ) Quantitative analysis of pDNA-PKcs s2056 by immunoblot analysis in (C). ( E ) U2OS cells were transfected with PP5, PP2A or PP1 siRNA for 72 h, then transfected with NHEJ reporter and/or I-SceI 4 h later, followed by treatment with or without CdCl 2 (100 μM) for 3 h; ChIP assay was performed using anti-DNA-PKcs antibody, followed by PCR to detect DNA-PKcs bound DSB fragment. ( F ) GC-2 cells stably expressing the PP5 wildtype (WT) or H304A mutant were treated by X-rays (2 Gy) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of the indicated protein in whole cells. * P < 0.05; ** P < 0.01; *** P < 0.001; ns indicates non-significant difference. Data presented as mean ± SD from three independent experiments.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Phospho-proteomics, Western Blot, Transfection, Stable Transfection, Expressing, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: PP5 activity was impaired by Cd ions in a competitive pattern. ( A ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) at the indicated times, followed by immunoblot analysis of PP5 on the chromatin fraction. ( B ) The dose effects of CdCl 2 on the activity of PP5 in vitro with Mn ions (0.2 μM) and Cd ions (0, 0.02, 0.04, 0.08, 0.2 μM). ( C ) GC-2 cells were treated by H 2 O 2 (40 μM) with or without CdCl 2 (20 μM) for 1 h, whole cell extracts were immunoprecipitated by PP5 antibody, and the activity of PP5 was determined in vitro . ( D ) The activity of PP5 inhibited by Cd ions (0.04 μM) was reversed by MnCl 2 (0, 0.04, 0.2, 0.4 μM), Zn (0, 0.04, 0.2, 0.4 μM) or Ca ions (0, 0.04, 0.2, 0.4 μM) in vitro . ( E ) GC-2 cells were treated by H 2 O 2 (40 μM) and CdCl 2 (20 μM) with or without MnCl 2 (10 μM) at the indicated times, followed by immunoblot analysis of indicated protein in whole cells. ( F ) U2OS cells were transfected with NHEJ reporter and I-SceI 4 h later, followed by treatment with CdCl 2 (100 μM) and with or without MnCl 2 (20 μM) for 3 h; ChIP assay was performed using anti-DNA-PKcs antibody, followed by PCR to detect DNA-PKcs bound DSB fragment. ( G ) GC-2 cells after transient cotransfection with the NHEJ reporter and/ or I-SceI, after which MnCl 2 (10 μM) was added 30 min before CdCl 2 (20 μM) treatment; the efficiency of NHEJ repair was determined by flow cytometry. n > 10 000 cells. ( H ) Representative images of the comet assay that was utilized to analyze the level of DNA damage in GC-2 cells, which were treated by H 2 O 2 (40 μM) with or without Cd ions (20 μM) and/or Mn ions (10 μM) at the indicated times. Scale bar, 100 μm. (I) Quantitative analysis of the DNA tail in cells treated as in (H). n > 100 cells.* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Data presented as mean ± SD.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Activity Assay, Western Blot, In Vitro, Immunoprecipitation, Transfection, Cotransfection, Flow Cytometry, Single Cell Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: Cd ions induced male reproductive dysfunction in a mouse model was reversed by Mn ions. ( A ) Representative images of the GC-2 cells treated by Cd ions (20 μM) for 24 h with or without Mn ions (10 μM), colony formation was determined. ( B ) Quantitative analysis of relative colony formation as in A, normalized to untreated group. ( C ) GC-2 cells were treated by Cd ions (20 μM) for 24 h, pretreated with or without Mn ions (10 μM), the cell viability was determined and normalized to the untreated group. ( D ) The percentage of motile sperm was determined after male mice intraperitoneal injection of Cd ions (2 mg/kg) with or without Mn ions (20 mg/kg). n = 8. ( E ) The percentage of abnormal sperm was determined after male mice were treated with Cd ions with or without Mn ions. n = 8. ( F ) Representative images of the immunohistochemistry of p-DNA-PKcs S2056 and γ-H2AX in testes of mice following intraperitoneal injection of Cd ions as pretreatment, with or without Mn ions. n = 8. Scale bar, 50 μm. ( G ) Quantitative analysis of p-DNA-PKcs S2056 expression in testis as indicated in (F). ( H ) Quantitative analysis of γ-H2AX expression in testis as indicated in (F). ** P < 0.01; *** P < 0.001. Data presented as mean ± SD.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Injection, Immunohistochemistry, Expressing

Journal: Nucleic Acids Research

Article Title: Heavy metal ions exchange driven protein phosphorylation cascade functions in genomic instability in spermatocytes and male infertility

doi: 10.1093/nar/gkad128

Figure Lengend Snippet: A model illustrating the process of Cd ions inhibited NHEJ repair pathway. In this model, Cd ions competitively replace activating ions (Mn) to inhibit the activity of PP5, which results in decreased dephosphorylation of DNA-PKcs at S2056 in its PQR cluster. Hyper-phosphorylation of DNA-PKcs leads to its disassociation from DNA ends and the Ku complex, impairing the followed DNA ends ligation.

Article Snippet: The antibodies used in this study were: phospho-H2AX (Ser139) (2577), phospho-H2AX (Ser139) (80312), H2AX (7631), phospho-CHK1 (Ser317) (12302), CHK1 (2360), CHK2 (6334), phospho-CHK2 (Thr68) (2197), Ku80 (2180), Ku70 (4588), phospho-DNA-PKcs (Ser2056) (68716), DNA-PKcs (38168), GAPDH (5174), PP2A (2038), Artemis (13381), ATM (2873), PP5 (2289), Histone H4 (13919), Phospho-ATM (Ser1981) (5883) and 53BP1 (88439), purchased from Cell Signaling Technology, USA; RAD51 (ab133534) and PP1 (ab134947), purchased from Abcam, USA; Phospho-DNA-PKcs (Thr2609) (NBP1-02456), purchased from NOVUS, USA.

Techniques: Activity Assay, De-Phosphorylation Assay, Phospho-proteomics, Ligation

Journal: Journal of Molecular and Cellular Cardiology Plus

Article Title: Transcriptomics reveal stretched human pluripotent stem cell-derived cardiomyocytes as an advantageous hypertrophy model

doi: 10.1016/j.jmccpl.2022.100020

Figure Lengend Snippet: Effects of p38 mitogen-activated protein kinase (p38 MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2) and protein kinase C (PKC) inhibitors on stretch-induced hypertrophic gene expression in human pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The following inhibitors were utilized: SB203580 at 10 μM to inhibit p38 MAPK, U0126 at 10 μM to inhibit MEK1/2, Gö6983 at 1 μM to inhibit all PKC isoforms and Gö6976 at 1 μM to inhibit classical PKC isoforms. Natriuretic peptide B ( NPPB ; A), skeletal muscle alpha actin 1 ( ACTA1 ; B), galanin ( GAL ; C) and long intergenic non-protein coding RNA 648 ( LINC00648 ; D) RNA expression was measured with qRT–PCR after a 24-h cyclic mechanical stretch. The results are presented as fold change relative to the unstretched control. The data are shown as mean ± standard error of the mean, and values from individual experiments are presented as dots ( n = 5, except for SB203580 and Gö6976 n = 4; where n represents biological replicates of cells from individual differentiations). * p < 0.05, ** p < 0.01 vs. unstretched DMSO, † p < 0.05 vs. stretched DMSO, Mann–Whitney U test.

Article Snippet: The mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 and the p38 MAPK inhibitor SB203580 were purchased from Tocris Bioscience (Bristol, UK), the pan-PKC inhibitor Gö6983 was purchased from STEMCELL Technologies (Vancouver, Canada) and the classical PKC inhibitor Gö6976 was purchased from Merck (Darmstadt, Germany).

Techniques: Gene Expression, Derivative Assay, RNA Expression, Quantitative RT-PCR, Control, MANN-WHITNEY

Journal: Frontiers in Oncology

Article Title: Suppression of EGFR/PKC-δ/NF-κB Signaling Associated With Imipramine-Inhibited Progression of Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2021.735183

Figure Lengend Snippet: The inhibition of EGFR/PKC-δ/NF-κB proteins phosphorylation and induction of apoptosis-related proteins by imipramine in CL1-5-F4/ NF-κB-luc2 bearing tumor. (A–C) The protein expression from IHC of EGFR (Try 1068), PKC-δ (Thr507), NF-κB (Ser536), cleaved caspase-3, -8, -9, MMP-9, XIAP, MCL-1 and their quantification bar chart are presented. (D, E) The tumor ex vivo Western blotting from each mice of cleaved caspase-3, -8, -9 and PARP-1 is presented. ( ** p < 0.01 vs . vehicle; scale bar =100 μm).

Article Snippet: Primary antibodies against Matrix metalloproteinase-9 (MMP-9) (AB19016, Millipore), vascular endothelial growth factor (VEGF) (ab1316, Abcam, Cambridge, UK), EGFR (Try 1068) (#2234, Cell signaling, Danvers, MA, USA), EGFR (E-AB-63555, Elabscience, Houston, TX, USA), PKC-δ (Thr507) (E-AB-20968, Elabscience), PKC-δ (E-AB-14675, Elabscience), NF-κB p65 (Ser536) (E-AB-70335, Elabscience), NF-κB p65 (E-AB-22066, Elabscience), cell leukemia-1 (MCL-1) (BV-438, BioVision), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) (D16A8, Cell signaling), X-linked inhibitor of apoptosis protein (XIAP) (PA5-29253, Thermo Fisher Scientific), Fas (E-AB-40063, Elabscience), Fas ligand (FasL) (E-AB-31410, Elabscience), cleaved caspase-3 (E-AB-30004, Elabscience), cleaved caspase-8 (E-AB-22107, Elabscience), cleaved caspase-9 (#9505, Cell Signaling Technology), PARP-1 (#9532, Cell Signaling Technology) and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, Waltam, MA, USA) for Western blotting were purchased from different companies as listed.

Techniques: Inhibition, Phospho-proteomics, Expressing, Ex Vivo, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling

doi: 10.3390/ijms241411842

Figure Lengend Snippet: ( A , B ): The protein levels of TLR2, TLR4, ß-catenin, and occludin in NPTr cells and TP pretreatment cells infection with APP. GAPDH in whole cell lysates was detected as the loading control. ( C ): the ratio of p-PKC-α/PKC-α, ( D ): the ratio of MLCK: Effects of TP on PKC and MLCK pathways in NPTrs activated by APP. ( E ): the ratio of p-JNK/JNK, ( F ): the ratio of p-p38/p38: Effect of TP on phospho-JNK, and -p38 and total -JNK, and -p38 expression in NPTrs by Western blotting. (mean ± SD, n = 3).* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The PVDF membranes were incubated overnight with the corresponding antibodies (Occludin Monoclonal antibody, Beta Catenin Monoclonal antibody, HRP-conjugated βeta actin antibody, TLR2 Monoclonal antibody, TLR4 Monoclonal antibody, MLCK Polyclonal antibody, JNK Monoclonal antibody, Phospho-JNK (Tyr185) Recombinant antibody, p38 MAPK Monoclonal antibody, and Phospho-p38 MAPK (Thr180/Tyr182) Polyclonal antibody, Proteintech, Chicago, IL, USA; PKC alpha Antibody, Phospho-PKC alpha (Ser657) Antibody, Affinity Biosciences, Cincinnati, OH, USA) at 4 °C.

Techniques: Infection, Control, Expressing, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

doi: 10.1128/mcb.21.21.7449-7459.2001

Figure Lengend Snippet: FIG. 1. Keratin-induced modulation of cell proliferation is related to signaling effectors belonging to the PI-3K pathway. (A) Keratin K10 (open bars) but not the NC form, which lacks amino and carboxyl termini (solid bars), inhibits cell growth in a variety of epithelial cell lines including human (HaCaT) and mouse (MCA3D and PB) keratinocytes and bovine mammary gland cells (BMGEH), but not in Rb-deficient human cervical carcinoma (C33A) cells. (B) Keratin K10 also mediates inhibition of HaCaT cell growth when coexpressed with human keratin K1, its natural partner in suprabasal, differentiating keratinocytes of the epidermis (see the introduction). (C) Keratin K10-induced HaCaT keratinocyte cell growth arrest is rescued by coexpression of Ha-ras, PDK1, Akt, and PKC but not by v-raf, RhoA, Rac1, Cdc42, or the activated catalytic subunit of PI-3K (p110CAAX). (D) Western blot using mouse MAb K8.60 (1/10,000 diluted) in lysates of cells cotransfected with K10 and the cited plasmids, demonstrating that the K10 expression levels in these transfections are similar. (E) Colony-forming experiments showing that Akt rescues

Article Snippet: Protein extracts (50 g in 500 l of phosphoprotein buffer) were incubated with agitation (overnight at 4°C) in the presence of 2 l of antibodies against K10 (mouse MAb K8.60), Akt and PKC (goat and rabbit polyclonal antibodies, respectively, from Santa Cruz Biotechnology), or anti-VSV-G tag (mouse MAb from Boehringer Mannheim).

Techniques: Inhibition, Western Blot, Expressing, Transfection

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

doi: 10.1128/mcb.21.21.7449-7459.2001

Figure Lengend Snippet: FIG. 3. Akt and PKC colocalize with the keratin cytoskeleton in cells transfected with K10 but not with a mutant form lacking both the amino and carboxyl termini (NC). HaCaT cells were transiently transfected with K10 (A and B) or NC (C and D) and analyzed by double immunofluorescence and confocal microscopy to visualize the transfected keratin (using the LH3 mouse MAb; A to D) and the endogenous Akt (using a goat polyclonal antibody; A and C) or PKC (using a rabbit polyclonal antibody; B and D). (E and F) Distribution of endogenous PKC (E) and Akt (F) in nontransfected HaCaT cells (see also arrows in panels A and B). Note that K10, but not NC, changes the distribution of the endogenous kinases from a dispersed to a filamentous shape, such that the distribution colocalizes with that of K10. Similar results were obtained with two different antibodies raised against different epitopes of Akt or PKC (not shown). Arrows (A and B), nontransfected cells. Representative examples from three independent experiments are shown. (G) Western blot using MAb LH3 demonstrating that K10 and NC are expressed to similar extents in transfected HaCaT cells (see also Fig. 7B). Bars, 10 m.

Article Snippet: Protein extracts (50 g in 500 l of phosphoprotein buffer) were incubated with agitation (overnight at 4°C) in the presence of 2 l of antibodies against K10 (mouse MAb K8.60), Akt and PKC (goat and rabbit polyclonal antibodies, respectively, from Santa Cruz Biotechnology), or anti-VSV-G tag (mouse MAb from Boehringer Mannheim).

Techniques: Transfection, Mutagenesis, Confocal Microscopy, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

doi: 10.1128/mcb.21.21.7449-7459.2001

Figure Lengend Snippet: FIG. 4. Impaired activation of PI-3K signaling effectors as a consequence of K10, but not NC, expression. HaCaT cells synchronized in G0 by serum starvation were transfected with K10 (A) or NC (B). After 48 h cells were allowed to reenter the cell cycle by serum addition. After 3 h, expression of the transfected construct (in red) and endogenous phosphorylated Akt (Akt-P; in green) was analyzed by indirect double immunofluorescence. The images shown are derived by merging the two immunofluorescence channels. Note that the expression of K10 leads to nondetectable Akt-P levels, whereas in cells transfected with NC at least some Akt-P (green and yellow) is present. (C and D) Keratin expression alters Akt (C) and PKC (D) activities. HaCaT cells were transfected with K10 or K16 and HA-tagged Akt (C) or PKC (D), and the kinase activities were determined following immunoprecipitation against the HA epitope using H2B and MBP, respectively, as substrates (see Materials and Methods). The lower portions of panels C and D show anti-HA tag immunoblots, demonstrating that similar amounts of immunoprecipitated Akt and PKC are found in the corresponding samples. Representative examples from three independent experiments are shown. Bars, 15 m.

Article Snippet: Protein extracts (50 g in 500 l of phosphoprotein buffer) were incubated with agitation (overnight at 4°C) in the presence of 2 l of antibodies against K10 (mouse MAb K8.60), Akt and PKC (goat and rabbit polyclonal antibodies, respectively, from Santa Cruz Biotechnology), or anti-VSV-G tag (mouse MAb from Boehringer Mannheim).

Techniques: Activation Assay, Expressing, Transfection, Construct, Derivative Assay, Immunoprecipitation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

doi: 10.1128/mcb.21.21.7449-7459.2001

Figure Lengend Snippet: FIG. 5. Akt and PKC interact with K10 during HaCaT keratinocyte differentiation. Spontaneously differentiated HaCaT keratinocytes (4, 27) were fixed and analyzed by indirect immunofluorescence to detect endogenous Akt (A) and keratin K10 (A). Note that in differentiating cells (arrow), characterized by the presence of K10, the Akt staining pattern changes, becoming filamentous and coincident with that of keratin K10 (A). (B) Akt and PKC are associated with keratins in differentiated HaCaT keratinocyte cultures. HaCaT cells were induced to differentiate by serum starvation for 12 days (22, 26, 27), and the soluble (Sol) and keratin-enriched fractions were obtained as described previously (25) and analyzed by Western blotting using antibodies against keratin K5, keratin K10, Akt, PKC, and Akt-P. Soluble and keratin-enriched fractions from control nondifferentiated cultures were analyzed in parallel. Note the presence of most Akt, PKC, and phosphorylated Akt in the soluble fraction in nondifferentiated cultures (K10 negative), whereas in differentiated cultures both Akt and PKC are in the insoluble (Insol)-keratin fraction, which contains keratin K10. The K5 signal demonstrates similar loadings in the samples from control and differentiating cells.

Article Snippet: Protein extracts (50 g in 500 l of phosphoprotein buffer) were incubated with agitation (overnight at 4°C) in the presence of 2 l of antibodies against K10 (mouse MAb K8.60), Akt and PKC (goat and rabbit polyclonal antibodies, respectively, from Santa Cruz Biotechnology), or anti-VSV-G tag (mouse MAb from Boehringer Mannheim).

Techniques: Staining, Western Blot, Control

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

doi: 10.1128/mcb.21.21.7449-7459.2001

Figure Lengend Snippet: FIG. 6. Distribution of Akt, Akt-P, and K10 in newborn mouse skin. (A to D) Frozen sections of newborn mouse skin were processed for immunofluorescence using antibodies against Akt (A), Akt-P (B), and K10 (C). The three immunofluorescence channels (Texas red, fluorescein isothiocyanate, and AMCA, respectively) are shown merged in panel D. Note that Akt is present throughout all the epidermal layers, whereas phosphorylated Akt is present exclusively in the basal layer and K10 is restricted to differentiated suprabasal layers. This shows that Akt and K10 are coexpressed in suprabasal cells but that only basal, K10-negative cells have active, phosphorylated Akt. Dashed lines, epidermal-dermal boundary. (E) Immunoprecipitation-Western blot experiments to biochemically confirm the K10-Akt-PKC interaction in skin. Whole-skin extracts were immunoprecipitated (IP) with the indicated antibodies and probed by Western blotting with the antibodies to proteins indicated at the right. Note the presence of K10, but not K5, in Akt and PKC immunoprecipitates and vice versa, whereas phosphorylated Akt is absent from K10 immunoprecipitates. Also note the absence of the kinases in K5 immunoprecipitates. Bar, 100 m.

Article Snippet: Protein extracts (50 g in 500 l of phosphoprotein buffer) were incubated with agitation (overnight at 4°C) in the presence of 2 l of antibodies against K10 (mouse MAb K8.60), Akt and PKC (goat and rabbit polyclonal antibodies, respectively, from Santa Cruz Biotechnology), or anti-VSV-G tag (mouse MAb from Boehringer Mannheim).

Techniques: Immunoprecipitation, Western Blot

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

doi: 10.1128/mcb.21.21.7449-7459.2001

Figure Lengend Snippet: FIG. 7. Expression of NTerm alleviates K10-induced cell growth arrest. (A) Colony-forming experiments with HaCaT cells demonstrate that the cotransfection of increasing amounts of an NTerm-encoding plasmid with 5 g of the K10-encoding plasmid rescues K10-promoted inhibition of cell proliferation. (B) Similar cotransfection experiments with C33A cells in the presence of co transfected pRb demonstrate that NTerm also restores transfected pRb phosphorylation and endogenous cyclin D1 levels. Lower two sections demonstrate that the levels of K10 and NC expression are similar and that the expression of increasing amounts of NTerm does not affect the expression of K10. Note also that, unlike K10, NC has no effect on pRb phophorylation and cyclin D1 levels. (C to D) Examples of triple immunofluorescence after transient cotransfection of 5 g of K10 and 5 g of NTerm in HaCaT cells, demonstrating that the colocalization of K10 and endogenous Akt and PKC is abolished in cells expressing NTerm. (C and D) Immunofluorescence visualizing K10. (C and D) Immunofluorescences visualizing Akt and PKC, respectively. (C and D ) Immunofluorescences visualizing NTerm. For comparison, see Fig. 3A, A, B, and B. Bars, (C and D), 15 m. Data in panel A are from triplicate independent experiments and are means SD.

Article Snippet: Protein extracts (50 g in 500 l of phosphoprotein buffer) were incubated with agitation (overnight at 4°C) in the presence of 2 l of antibodies against K10 (mouse MAb K8.60), Akt and PKC (goat and rabbit polyclonal antibodies, respectively, from Santa Cruz Biotechnology), or anti-VSV-G tag (mouse MAb from Boehringer Mannheim).

Techniques: Expressing, Cotransfection, Plasmid Preparation, Inhibition, Transfection, Phospho-proteomics, Comparison

Journal: Molecular and Cellular Biology

Article Title: Inhibition of Protein Kinase B (PKB) and PKCζ Mediates Keratin K10-Induced Cell Cycle Arrest

doi: 10.1128/mcb.21.21.7449-7459.2001

Figure Lengend Snippet: FIG. 8. NTerm interacts with Akt in vitro and in vivo. (A) Immunoprecipitation using an anti-VSV-G MAb of extracts from pooled clones of VSV-G-tagged NTerm- and vector-transfected HaCaT cells, followed by Western blotting against VSV-G, Akt, and PKC showing that both Akt and PKC coprecipitate with NTerm. (B) Two-cell hybrid experiments using the ras rescue system (6). cdc25-2 yeast cells were cotransformed with the indicated plasmids and plated at the permissive (25°C) or the restrictive (37°C) temperature onto galactose-containing plates. Note that, besides the positive controls including RasCAAX expression (1 and 3) and pMyrMAFB plus pSOSMAFB expression (6), growth at the restrictive temperature was obtained with the cotransformation of pMyrAkt plus RasNTerm (4), indicating that protein-protein interaction between myrAkt and NTerm has occurred. The absence of growth in 2 demonstrates that NTerm by itself cannot override the mutation of cdc25-2 yeast cells. As a negative control, a myristoylated form of phosduccin D (PhosD) was cotransformed with pRasNTerm (5). No colonies were observed at 37°C in glucose-containing, galactose-depleted plates, indicating that myrAkt alone does not allow yeast cell growth (not shown).

Article Snippet: Protein extracts (50 g in 500 l of phosphoprotein buffer) were incubated with agitation (overnight at 4°C) in the presence of 2 l of antibodies against K10 (mouse MAb K8.60), Akt and PKC (goat and rabbit polyclonal antibodies, respectively, from Santa Cruz Biotechnology), or anti-VSV-G tag (mouse MAb from Boehringer Mannheim).

Techniques: In Vitro, In Vivo, Immunoprecipitation, Clone Assay, Plasmid Preparation, Transfection, Western Blot, Expressing, Mutagenesis, Negative Control