phospho pkc pan βii ser660 cell signaling technology frankfurt germany  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho pkc pan βii ser660 cell signaling technology frankfurt germany
    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII <t>Ser660)</t> in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.
    Phospho Pkc Pan βii Ser660 Cell Signaling Technology Frankfurt Germany, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Anti-Inflammatory Effects of GPR55 Agonists and Antagonists in LPS-Treated BV2 Microglial Cells"

    Article Title: Anti-Inflammatory Effects of GPR55 Agonists and Antagonists in LPS-Treated BV2 Microglial Cells

    Journal: Pharmaceuticals

    doi: 10.3390/ph17060674

    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.
    Figure Legend Snippet: Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.

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    rabbit anti phospho pkc pan  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho pkc pan
    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of <t>PKC</t> (pan) <t>(βII</t> <t>Ser660)</t> in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.
    Rabbit Anti Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Anti-Inflammatory Effects of GPR55 Agonists and Antagonists in LPS-Treated BV2 Microglial Cells"

    Article Title: Anti-Inflammatory Effects of GPR55 Agonists and Antagonists in LPS-Treated BV2 Microglial Cells

    Journal: Pharmaceuticals

    doi: 10.3390/ph17060674

    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.
    Figure Legend Snippet: Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.

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    rabbit anti phospho pkc pan  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho pkc pan
    Rabbit Anti Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit anti phospho pkc pan  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit anti phospho pkc pan
    Effects of X15856 on the phosphorylation of <t>PKC</t> (pan) <t>(βII</t> <t>Ser660)</t> ( A ) and NF-κB ( B ) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Material and Methods Section. Values are presented as the mean ± SD of at least three independent experiments, and protein levels were referenced to Vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05 compared to LPS.
    Rabbit Anti Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 86 stars, based on 1 article reviews
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    rabbit anti phospho pkc pan - by Bioz Stars, 2024-07
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    1) Product Images from "Anti-Neuroinflammatory Effects of a Macrocyclic Peptide-Peptoid Hybrid in Lipopolysaccharide-Stimulated BV2 Microglial Cells"

    Article Title: Anti-Neuroinflammatory Effects of a Macrocyclic Peptide-Peptoid Hybrid in Lipopolysaccharide-Stimulated BV2 Microglial Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms25084462

    Effects of X15856 on the phosphorylation of PKC (pan) (βII Ser660) ( A ) and NF-κB ( B ) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Material and Methods Section. Values are presented as the mean ± SD of at least three independent experiments, and protein levels were referenced to Vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05 compared to LPS.
    Figure Legend Snippet: Effects of X15856 on the phosphorylation of PKC (pan) (βII Ser660) ( A ) and NF-κB ( B ) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Material and Methods Section. Values are presented as the mean ± SD of at least three independent experiments, and protein levels were referenced to Vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05 compared to LPS.

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    phospho pkc pan  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho pkc pan
    MAPK15 induces <t>PKC-dependent</t> <t>NRF2</t> Serine 40 phosphorylation and protein stabilization. ( A ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( B ) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H 2 O 2 , for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( C ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( D ) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H 2 O 2 for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( E ) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H 2 O 2 , for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. ( F ) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H 2 O 2 or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( G ) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti -MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. ( H ) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.
    Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "MAPK15 controls cellular responses to oxidative stress by regulating NRF2 activity and expression of its downstream target genes"

    Article Title: MAPK15 controls cellular responses to oxidative stress by regulating NRF2 activity and expression of its downstream target genes

    Journal: Redox Biology

    doi: 10.1016/j.redox.2024.103131

    MAPK15 induces PKC-dependent NRF2 Serine 40 phosphorylation and protein stabilization. ( A ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( B ) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H 2 O 2 , for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( C ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( D ) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H 2 O 2 for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( E ) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H 2 O 2 , for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. ( F ) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H 2 O 2 or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( G ) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti -MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. ( H ) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.
    Figure Legend Snippet: MAPK15 induces PKC-dependent NRF2 Serine 40 phosphorylation and protein stabilization. ( A ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( B ) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H 2 O 2 , for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( C ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( D ) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H 2 O 2 for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( E ) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H 2 O 2 , for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. ( F ) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H 2 O 2 or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( G ) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti -MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. ( H ) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.

    Techniques Used: Transfection, Plasmid Preparation, SDS Page, Immunofluorescence, Fluorescence, Microscopy, Immunoprecipitation, Expressing, Luciferase, Activity Assay

    anti phospho pkc pan  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho pkc pan
    Anti Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    rabbit pab anti phospho pkc pan  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit pab anti phospho pkc pan

    Rabbit Pab Anti Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting"

    Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting

    Journal: Cell reports

    doi: 10.1016/j.celrep.2024.113700


    Figure Legend Snippet:

    Techniques Used: Virus, Recombinant, Modification, Saline, Protease Inhibitor, Marker, Cytotoxicity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Knock-Out, Software

    pan phospho pkc  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc pan phospho pkc
    PKCs are involved in tight junction regulation in the absence of MUC13. (A) Immunoblot analysis of PKCα, PKCβ-I, PKCδ, <t>PKCε,</t> <t>PKCζ,</t> <t>phospho-PKC</t> (pan) and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (B) Quantification of relative protein expression of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ and phospho-PKC (pan) shown in panel A. For some of the replicate experiments performed for Fig. 8A,B, the same loading control was used as replicates of experiments depicted in <xref ref-type=Fig. 7C,D . (C) Overlay of fluorescence images for MUC13-CT (red), PKCδ (green) and nuclei (white) in HRT18 cells grown for 2 weeks with orthogonal views. The yellow arrow indicates positions in which staining for both proteins can be observed in close proximity. Scale bar: 20 μm. (D) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 µM PKC inhibitor (GF-109203X) added every 3 days. Immunoblots for claudin-1, claudin-3, claudin-4 and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (E) Quantification of relative protein expression of claudin-1, claudin-3 and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in panel D. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and s.e.m. of three independent experiments. Statistical analysis for B and E was conducted using one-sample two-tailed unpaired t -tests after normalizing to the untreated (no inhibitor) sample. ns, non-significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Schematic model depicting tight junction regulation by MUC13. In WT cells (left panel), MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs and E-cadherin are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. The cytoplasmic tail of MUC13 has a putative PKC-binding motif, which might play a role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin and ZO-1 can also be targeted by PKCs. In MUC13 knockout cells (middle panel), TJ proteins (occludin, claudins and ZO-1) accumulate at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. MUC13-ΔCT cells (right panel) have an intermediate phenotype with some accumulation of claudin-1, -3 and -4 and ZO-1 at the membrane, but to a lower extent compared to that in the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to that in WT and ΔMUC13 cells. " width="250" height="auto" />
    Pan Phospho Pkc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C"

    Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.261468

    PKCs are involved in tight junction regulation in the absence of MUC13. (A) Immunoblot analysis of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ, phospho-PKC (pan) and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (B) Quantification of relative protein expression of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ and phospho-PKC (pan) shown in panel A. For some of the replicate experiments performed for Fig. 8A,B, the same loading control was used as replicates of experiments depicted in <xref ref-type=Fig. 7C,D . (C) Overlay of fluorescence images for MUC13-CT (red), PKCδ (green) and nuclei (white) in HRT18 cells grown for 2 weeks with orthogonal views. The yellow arrow indicates positions in which staining for both proteins can be observed in close proximity. Scale bar: 20 μm. (D) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 µM PKC inhibitor (GF-109203X) added every 3 days. Immunoblots for claudin-1, claudin-3, claudin-4 and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (E) Quantification of relative protein expression of claudin-1, claudin-3 and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in panel D. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and s.e.m. of three independent experiments. Statistical analysis for B and E was conducted using one-sample two-tailed unpaired t -tests after normalizing to the untreated (no inhibitor) sample. ns, non-significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Schematic model depicting tight junction regulation by MUC13. In WT cells (left panel), MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs and E-cadherin are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. The cytoplasmic tail of MUC13 has a putative PKC-binding motif, which might play a role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin and ZO-1 can also be targeted by PKCs. In MUC13 knockout cells (middle panel), TJ proteins (occludin, claudins and ZO-1) accumulate at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. MUC13-ΔCT cells (right panel) have an intermediate phenotype with some accumulation of claudin-1, -3 and -4 and ZO-1 at the membrane, but to a lower extent compared to that in the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to that in WT and ΔMUC13 cells. " title="... Immunoblot analysis of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ, phospho-PKC (pan) and β-actin in total lysates of monolayers ..." property="contentUrl" width="100%" height="100%"/>
    Figure Legend Snippet: PKCs are involved in tight junction regulation in the absence of MUC13. (A) Immunoblot analysis of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ, phospho-PKC (pan) and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (B) Quantification of relative protein expression of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ and phospho-PKC (pan) shown in panel A. For some of the replicate experiments performed for Fig. 8A,B, the same loading control was used as replicates of experiments depicted in Fig. 7C,D . (C) Overlay of fluorescence images for MUC13-CT (red), PKCδ (green) and nuclei (white) in HRT18 cells grown for 2 weeks with orthogonal views. The yellow arrow indicates positions in which staining for both proteins can be observed in close proximity. Scale bar: 20 μm. (D) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 µM PKC inhibitor (GF-109203X) added every 3 days. Immunoblots for claudin-1, claudin-3, claudin-4 and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (E) Quantification of relative protein expression of claudin-1, claudin-3 and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in panel D. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and s.e.m. of three independent experiments. Statistical analysis for B and E was conducted using one-sample two-tailed unpaired t -tests after normalizing to the untreated (no inhibitor) sample. ns, non-significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Schematic model depicting tight junction regulation by MUC13. In WT cells (left panel), MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs and E-cadherin are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. The cytoplasmic tail of MUC13 has a putative PKC-binding motif, which might play a role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin and ZO-1 can also be targeted by PKCs. In MUC13 knockout cells (middle panel), TJ proteins (occludin, claudins and ZO-1) accumulate at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. MUC13-ΔCT cells (right panel) have an intermediate phenotype with some accumulation of claudin-1, -3 and -4 and ZO-1 at the membrane, but to a lower extent compared to that in the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to that in WT and ΔMUC13 cells.

    Techniques Used: Western Blot, Expressing, Fluorescence, Staining, Isolation, Membrane, Two Tailed Test, Binding Assay, Activity Assay, Knock-Out

    rrid ab 10694715 rabbit pab anti phospho pkc pan cell signaling 9371  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho pkc pan βii ser660 cell signaling technology frankfurt germany
    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII <t>Ser660)</t> in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.
    Phospho Pkc Pan βii Ser660 Cell Signaling Technology Frankfurt Germany, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit anti phospho pkc pan
    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of <t>PKC</t> (pan) <t>(βII</t> <t>Ser660)</t> in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.
    Rabbit Anti Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc phospho pkc pan
    MAPK15 induces <t>PKC-dependent</t> <t>NRF2</t> Serine 40 phosphorylation and protein stabilization. ( A ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( B ) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H 2 O 2 , for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( C ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( D ) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H 2 O 2 for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( E ) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H 2 O 2 , for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. ( F ) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H 2 O 2 or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( G ) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti -MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. ( H ) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.
    Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho pkc pan
    MAPK15 induces <t>PKC-dependent</t> <t>NRF2</t> Serine 40 phosphorylation and protein stabilization. ( A ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( B ) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H 2 O 2 , for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( C ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( D ) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H 2 O 2 for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( E ) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H 2 O 2 , for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. ( F ) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H 2 O 2 or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( G ) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti -MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. ( H ) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.
    Anti Phospho Pkc Pan, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rabbit pab anti phospho pkc pan

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    Cell Signaling Technology Inc pan phospho pkc
    PKCs are involved in tight junction regulation in the absence of MUC13. (A) Immunoblot analysis of PKCα, PKCβ-I, PKCδ, <t>PKCε,</t> <t>PKCζ,</t> <t>phospho-PKC</t> (pan) and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (B) Quantification of relative protein expression of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ and phospho-PKC (pan) shown in panel A. For some of the replicate experiments performed for Fig. 8A,B, the same loading control was used as replicates of experiments depicted in <xref ref-type=Fig. 7C,D . (C) Overlay of fluorescence images for MUC13-CT (red), PKCδ (green) and nuclei (white) in HRT18 cells grown for 2 weeks with orthogonal views. The yellow arrow indicates positions in which staining for both proteins can be observed in close proximity. Scale bar: 20 μm. (D) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 µM PKC inhibitor (GF-109203X) added every 3 days. Immunoblots for claudin-1, claudin-3, claudin-4 and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (E) Quantification of relative protein expression of claudin-1, claudin-3 and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in panel D. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and s.e.m. of three independent experiments. Statistical analysis for B and E was conducted using one-sample two-tailed unpaired t -tests after normalizing to the untreated (no inhibitor) sample. ns, non-significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Schematic model depicting tight junction regulation by MUC13. In WT cells (left panel), MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs and E-cadherin are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. The cytoplasmic tail of MUC13 has a putative PKC-binding motif, which might play a role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin and ZO-1 can also be targeted by PKCs. In MUC13 knockout cells (middle panel), TJ proteins (occludin, claudins and ZO-1) accumulate at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. MUC13-ΔCT cells (right panel) have an intermediate phenotype with some accumulation of claudin-1, -3 and -4 and ZO-1 at the membrane, but to a lower extent compared to that in the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to that in WT and ΔMUC13 cells. " width="250" height="auto" />
    Pan Phospho Pkc, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc rrid ab 10694715 rabbit pab anti phospho pkc pan cell signaling 9371
    PKCs are involved in tight junction regulation in the absence of MUC13. (A) Immunoblot analysis of PKCα, PKCβ-I, PKCδ, <t>PKCε,</t> <t>PKCζ,</t> <t>phospho-PKC</t> (pan) and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (B) Quantification of relative protein expression of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ and phospho-PKC (pan) shown in panel A. For some of the replicate experiments performed for Fig. 8A,B, the same loading control was used as replicates of experiments depicted in <xref ref-type=Fig. 7C,D . (C) Overlay of fluorescence images for MUC13-CT (red), PKCδ (green) and nuclei (white) in HRT18 cells grown for 2 weeks with orthogonal views. The yellow arrow indicates positions in which staining for both proteins can be observed in close proximity. Scale bar: 20 μm. (D) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 µM PKC inhibitor (GF-109203X) added every 3 days. Immunoblots for claudin-1, claudin-3, claudin-4 and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (E) Quantification of relative protein expression of claudin-1, claudin-3 and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in panel D. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and s.e.m. of three independent experiments. Statistical analysis for B and E was conducted using one-sample two-tailed unpaired t -tests after normalizing to the untreated (no inhibitor) sample. ns, non-significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Schematic model depicting tight junction regulation by MUC13. In WT cells (left panel), MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs and E-cadherin are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. The cytoplasmic tail of MUC13 has a putative PKC-binding motif, which might play a role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin and ZO-1 can also be targeted by PKCs. In MUC13 knockout cells (middle panel), TJ proteins (occludin, claudins and ZO-1) accumulate at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. MUC13-ΔCT cells (right panel) have an intermediate phenotype with some accumulation of claudin-1, -3 and -4 and ZO-1 at the membrane, but to a lower extent compared to that in the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to that in WT and ΔMUC13 cells. " width="250" height="auto" />
    Rrid Ab 10694715 Rabbit Pab Anti Phospho Pkc Pan Cell Signaling 9371, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.

    Journal: Pharmaceuticals

    Article Title: Anti-Inflammatory Effects of GPR55 Agonists and Antagonists in LPS-Treated BV2 Microglial Cells

    doi: 10.3390/ph17060674

    Figure Lengend Snippet: Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.

    Article Snippet: The antibody used for PKC detection in the current study was phospho-PKC (pan) (βII Ser660) (Cell Signaling Technology, Frankfurt, Germany), detecting PKC α, βI, βII, δ, ε, η and θ isoforms when phosphorylated at a carboxy-terminal residue homologous to serine 660 of PKC βII (Cell Signaling Technology, Frankfurt, Germany).

    Techniques:

    Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.

    Journal: Pharmaceuticals

    Article Title: Anti-Inflammatory Effects of GPR55 Agonists and Antagonists in LPS-Treated BV2 Microglial Cells

    doi: 10.3390/ph17060674

    Figure Lengend Snippet: Effects of KIT C ( A ), ML-193 ( B ), and O-1602 ( C ) on phosphorylation of PKC (pan) (βII Ser660) in LPS-stimulated BV2 cells. Cells were stimulated as described in the Materials and Methods section (30 min stimulation). Values are presented as the mean ± SD of at least three independent experiments (N = 3 ( A , B ), N = 4 ( C )), and protein levels were referenced to vinculin. Statistical analysis was performed using one-way ANOVA with Dunnett’s post hoc tests with * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to LPS-stimulated cells. The graphs show an average of the biological replicates and do not directly represent the blot shown above.

    Article Snippet: Primary antibodies were rabbit anti-phospho-PKC (pan) (βII Ser660) (cat. no.: 9371S, 1:1000; Cell Signaling Technology, Frankfurt, Germany), rabbit anti-ERK1/2 (cat. no.: 9101S, 1:1000; Cell Signaling Technology, Frankfurt, Germany), rabbit anti-p38 MAPK (cat. no.: 9211S, 1:1000; Cell Signaling Technology), rabbit anti-phospho-NF-kappaB-p65 (cat. No.: 3031S, 1:1000; Cell Signaling Technology, Frankfurt, Germany), and mouse anti-Vinculin (cat. no.: V9264, 1:20,000, Sigma-Aldrich GmbH, Taufkirchen, Germany).

    Techniques:

    MAPK15 induces PKC-dependent NRF2 Serine 40 phosphorylation and protein stabilization. ( A ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( B ) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H 2 O 2 , for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( C ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( D ) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H 2 O 2 for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( E ) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H 2 O 2 , for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. ( F ) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H 2 O 2 or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( G ) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti -MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. ( H ) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.

    Journal: Redox Biology

    Article Title: MAPK15 controls cellular responses to oxidative stress by regulating NRF2 activity and expression of its downstream target genes

    doi: 10.1016/j.redox.2024.103131

    Figure Lengend Snippet: MAPK15 induces PKC-dependent NRF2 Serine 40 phosphorylation and protein stabilization. ( A ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG plus empty vector or MAPK15_WT. After 24 h, they were treated with 50 μM Cycloheximide (CHX), for indicated times. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( B ) 293T cells were transiently co-overexpressed with NRF2-MYC-FLAG plus empty vector or MAPK15_WT or MAPK15_KD. After 24 h, samples were treated with 300 μM H 2 O 2 , for 1 h, and the lysates were subjected to SDS-PAGE followed by WB and analyzed for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( C ) 293T cells were transiently co-transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG S40A, plus empty vector or MAPK15_WT. After 24 h. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( D ) 293T cells were transfected with siSCR or siMAPK15. Then, after 24 h, they were transfected with NRF2-MYC-FLAG. After additional 48 h, cells were treated with 300 μM H 2 O 2 for 1 h and lysates were analyzed by WB for indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( E ) 293T cells were transfected with siSCR or siMAPK15 and, after 24 h, transfected with empty vector or NRF2-MYC-FLAG. After a total of 72h, samples were treated with 300 μM H 2 O 2 , for 1 h, then fixed and subjected to immunofluorescence analysis. Scale bars correspond to 10 μm. The accompanied graph shows intensitometric analysis of phospho-NRF2 S40A fluorescence from five representative microscopy fields signal per cell ± SD. ( F ) 293T cells were transiently transfected with empty vector or MAPK15_WT or treated with 300 μM H 2 O 2 or 200 nM TPA for 1 h, as positive controls. Lysates were analyzed by WB for the indicated proteins. One experiment, representative of 3 independent experiments, is shown. Densitometric analysis of bands is indicated. ( G ) 293T cells were transiently transfected with NRF2-MYC-FLAG or NRF2-MYC-FLAG_S40A plus empty vector or HA-MAPK15_WT. After 24 h, samples were treated, as indicated, with 10 μM GO6983 for 2 h. Lysates were next immunoprecipitated with anti -MYC antibodies and subjected to SDS-PAGE followed by WB to detect phospho-NRF2-S40 protein. Total lysates were also analyzed for expression of indicated proteins. One experiment, representative of 3 independent experiments, is shown. ( H ) 293T cells were transfected with ARE luciferase reporter vector plus empty vector or MAPK15_WT. Twenty-four hours after transfection, samples were treated with 5 μM or 10 μM GO6983, for 2 h. Then, samples were lysed, and luciferase activity was measured in cell extracts. Data are represented as fold induction of the normalized luciferase activity compared to control cells transfected with GFP. All luciferase results represent the average ± S.D. of three independent experiments. All samples were read in triplicate.

    Article Snippet: The following primary antibodies were used for western blots and confocal microscopy experiments: anti -MAPK15 (Invitrogen PA5-75930), anti-HA (Covance, MMS-101R) anti -MAPK1 (Santa Cruz, sc-154), anti -NRF2 (Cell Signaling, 12721), anti -phospho-NRF2_Ser40 (Abcam, ab180844), anti -IKBα (Santa Cruz, sc-371), anti-Flag (Sigma, F1804) anti -γH2A.X (Cell Signaling, 9718), anti-53BP1 (Novus, NB100-304), anti -NQO1 (Santa Cruz, sc-207495A), anti–HO–1 (Santa Cruz, sc-136960 for mouse samples and Genetex GTX101147 for human samples), anti -phospho-NRF2_Ser40 (Invitrogen, PA567520), anti -SLC7A11 (Cell Signaling, D2M7A); Anti -Phospho-PKC (pan) (βII Ser660) (Cell Signaling, 9371); anti -PKC (A-3) (Santa Cruz, sc-17769); anti -Myc (Cell Signaling, 2276); anti -Phospho-Ser/Thr-Pro (Upstate, 05–368).

    Techniques: Transfection, Plasmid Preparation, SDS Page, Immunofluorescence, Fluorescence, Microscopy, Immunoprecipitation, Expressing, Luciferase, Activity Assay

    Journal: Cell reports

    Article Title: Arachidonic acid inhibition of the NLRP3 inflammasome is a mechanism to explain the anti-inflammatory effects of fasting

    doi: 10.1016/j.celrep.2024.113700

    Figure Lengend Snippet:

    Article Snippet: Rabbit pAb anti-phospho-PKC (pan) , Cell Signaling , 9371; RRID AB_2168219.

    Techniques: Virus, Recombinant, Modification, Saline, Protease Inhibitor, Marker, Cytotoxicity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Knock-Out, Software

    PKCs are involved in tight junction regulation in the absence of MUC13. (A) Immunoblot analysis of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ, phospho-PKC (pan) and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (B) Quantification of relative protein expression of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ and phospho-PKC (pan) shown in panel A. For some of the replicate experiments performed for Fig. 8A,B, the same loading control was used as replicates of experiments depicted in <xref ref-type=Fig. 7C,D . (C) Overlay of fluorescence images for MUC13-CT (red), PKCδ (green) and nuclei (white) in HRT18 cells grown for 2 weeks with orthogonal views. The yellow arrow indicates positions in which staining for both proteins can be observed in close proximity. Scale bar: 20 μm. (D) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 µM PKC inhibitor (GF-109203X) added every 3 days. Immunoblots for claudin-1, claudin-3, claudin-4 and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (E) Quantification of relative protein expression of claudin-1, claudin-3 and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in panel D. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and s.e.m. of three independent experiments. Statistical analysis for B and E was conducted using one-sample two-tailed unpaired t -tests after normalizing to the untreated (no inhibitor) sample. ns, non-significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Schematic model depicting tight junction regulation by MUC13. In WT cells (left panel), MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs and E-cadherin are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. The cytoplasmic tail of MUC13 has a putative PKC-binding motif, which might play a role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin and ZO-1 can also be targeted by PKCs. In MUC13 knockout cells (middle panel), TJ proteins (occludin, claudins and ZO-1) accumulate at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. MUC13-ΔCT cells (right panel) have an intermediate phenotype with some accumulation of claudin-1, -3 and -4 and ZO-1 at the membrane, but to a lower extent compared to that in the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to that in WT and ΔMUC13 cells. " width="100%" height="100%">

    Journal: Journal of Cell Science

    Article Title: MUC13 negatively regulates tight junction proteins and intestinal epithelial barrier integrity via protein kinase C

    doi: 10.1242/jcs.261468

    Figure Lengend Snippet: PKCs are involved in tight junction regulation in the absence of MUC13. (A) Immunoblot analysis of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ, phospho-PKC (pan) and β-actin in total lysates of monolayers grown for 2 weeks. Molecular mass standards (kDa) are indicated on the left. (B) Quantification of relative protein expression of PKCα, PKCβ-I, PKCδ, PKCε, PKCζ and phospho-PKC (pan) shown in panel A. For some of the replicate experiments performed for Fig. 8A,B, the same loading control was used as replicates of experiments depicted in Fig. 7C,D . (C) Overlay of fluorescence images for MUC13-CT (red), PKCδ (green) and nuclei (white) in HRT18 cells grown for 2 weeks with orthogonal views. The yellow arrow indicates positions in which staining for both proteins can be observed in close proximity. Scale bar: 20 μm. (D) Immunoblot analysis of isolated membrane fractions from monolayers grown for 2 weeks in the presence/absence of 20 µM PKC inhibitor (GF-109203X) added every 3 days. Immunoblots for claudin-1, claudin-3, claudin-4 and the control protein Na + /K + -ATPase are shown. Molecular mass standards (kDa) are indicated on the left. (E) Quantification of relative protein expression of claudin-1, claudin-3 and claudin-4 in isolated fractions of cells grown in the presence/absence of GF-109203X as depicted in panel D. All assays were performed at least three times and representative images are shown. One representative clone for each cell line was used in these experiments. Bars represent average and s.e.m. of three independent experiments. Statistical analysis for B and E was conducted using one-sample two-tailed unpaired t -tests after normalizing to the untreated (no inhibitor) sample. ns, non-significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Schematic model depicting tight junction regulation by MUC13. In WT cells (left panel), MUC13 localizes to both the apical surface and tight junction (TJ) region of the lateral membrane. Cell junction complexes that contain claudins, occludin, ZOs and E-cadherin are assembled along the lateral membrane. Under normal conditions, there is some paracellular passage of ions and small solutes, a process that is controlled by the TJ proteins claudins and occludin. The cytoplasmic tail of MUC13 has a putative PKC-binding motif, which might play a role in recruiting PKC and controlling its activity and/or stability. Cell junction proteins such as claudins, occludin and ZO-1 can also be targeted by PKCs. In MUC13 knockout cells (middle panel), TJ proteins (occludin, claudins and ZO-1) accumulate at the membrane over time, causing increased transepithelial resistance (TEER) and lower paracellular passage of small solutes. The TEER buildup in ΔMUC13 cells is dependent on MLCK, ROCK and PKC kinases. The accumulation of claudins at the membrane in ΔMUC13 cells is PKC dependent and is not caused by slower degradation rates of TJ proteins through recycling endosomes. MUC13-ΔCT cells (right panel) have an intermediate phenotype with some accumulation of claudin-1, -3 and -4 and ZO-1 at the membrane, but to a lower extent compared to that in the full knockout. The role of PKC in this cell line remains to be determined. MUC13-ΔCT cells are less permeable to small solutes but do not show a significant increase in TEER compared to WT cells. The degradation rate of TJ proteins in MUC13-ΔCT cells is comparable to that in WT and ΔMUC13 cells.

    Article Snippet: For western blotting (WB) and immunofluorescence (IF), antibodies against claudin-1 (Thermo Fisher Scientific, 51-9000, 1:500 for WB), claudin-3 (Thermo Fisher Scientific, 34-1700, 1:500 for WB), claudin-4 (Thermo Fisher Scientific, 32-9400, 1:500 for WB, 1:50 for IF), occludin (Invitrogen, 33-1500, 1:500 for WB, 1:50 for IF), E-cadherin (Abcam, ab1416, 1:1000 for WB, 1:100 for IF), PKCα (Abcam, ab32376, 1:500 for WB), PKCβ-I (Abcam, ab195039, 1:500 for WB), PKCδ (Abcam, ab182126, 1:500 for WB; BD biosciences 610397, 1:100 for IF), PKCε (Abcam, ab63638, 1:500 for WB), PKCζ (Santa Cruz Biotechnology, sc-216, 1:500 for WB), pan phospho-PKC (βII Ser660) (Cell Signaling Technology, 9371, 1:500 for WB), GAPDH (Merck, G9545, 1:1000 for WB), β-actin (Bioss, bs-0061R, 1:2000 for WB, 1:100 for IF), MUC13 (specific for the cytoplasmic tail; Abcam, ab235450, 1:1000 for WB, 1:100 for IF), MUC13 hybridoma supernatant (specific for the extracellular domain; in house, see below for details, undiluted for IF), MAL-II-biotinylated lectin (Vector Lab, B-1265-1, 1:100 for IF), Na + /K + -ATPase (Abcam, ab76020, 1:1000 for WB), ZO-1 (rabbit, Abcam, ab216880, 1:100 for IF) and acetyl-histone H3 at K9 (Merck, 07-352, 1:1000 for WB) were used.

    Techniques: Western Blot, Expressing, Fluorescence, Staining, Isolation, Membrane, Two Tailed Test, Binding Assay, Activity Assay, Knock-Out