anti phospho rxx s t antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho rxx s t antibody
    hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif <t>RXX(s/t).</t> b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.
    Anti Phospho Rxx S T Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Fibroblast Growth Factor 21 Improves Insulin Sensitivity and Synergizes with Insulin in Human Adipose Stem Cell-Derived (hASC) Adipocytes"

    Article Title: Fibroblast Growth Factor 21 Improves Insulin Sensitivity and Synergizes with Insulin in Human Adipose Stem Cell-Derived (hASC) Adipocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111767

    hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif RXX(s/t). b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.
    Figure Legend Snippet: hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif RXX(s/t). b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.

    Techniques Used: Western Blot

    anti phospho rxx s t antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho rxx s t antibody
    hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif <t>RXX(s/t).</t> b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.
    Anti Phospho Rxx S T Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti phospho rxx s t antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Fibroblast Growth Factor 21 Improves Insulin Sensitivity and Synergizes with Insulin in Human Adipose Stem Cell-Derived (hASC) Adipocytes"

    Article Title: Fibroblast Growth Factor 21 Improves Insulin Sensitivity and Synergizes with Insulin in Human Adipose Stem Cell-Derived (hASC) Adipocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0111767

    hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif RXX(s/t). b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.
    Figure Legend Snippet: hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif RXX(s/t). b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.

    Techniques Used: Western Blot

    phosphorylated akt substrate motif specific antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phosphorylated akt substrate motif specific antibodies
    Effect of IGF-1 on <t>AKT/GSK-3β</t> activity <t>and</t> <t>cyclin</t> D1 expression in mesangial cells. (A) Mesangial cells were grown to 60-70% confluence, serum-starved overnight and treated with IGF-1 (100 ng/ml) for 1-5 min with or without 100 nM Wortmannin. Cell extracts (500 μg protein) were immunoprecipitated with anti-Akt antibodies and kinase activity was determined by using an Akt/PKB assay kit from Upstate Biotechnology. The basal Akt activity was ~4.8 pmol 32 Pi incorporated into Akt peptides/0.5 mg protein/10 min (n=5). (B) Mesangial cells were serum-starved overnight and incubated with IGF-1 (100 ng/ml) for different time periods: 0, 1, 3, 5, and 10 min, respectively, and 30 μg proteins were subjected to Western Blotting with anti-phospho-GSK-3β antibodies (n=3). The membrane was stripped and reprobed with anti-GSK-3α/β antibodies. (C) Mesangial cells were treated with IGF-1 (100 ng/ml) for 0, 2, 6 and 12 h and were subjected to Western blotting for cyclin D1 and reprobed for α-tubulin (a representative of n=3).
    Phosphorylated Akt Substrate Motif Specific Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells"

    Article Title: Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

    Journal: International Journal of Biological Sciences

    doi:

    Effect of IGF-1 on AKT/GSK-3β activity and cyclin D1 expression in mesangial cells. (A) Mesangial cells were grown to 60-70% confluence, serum-starved overnight and treated with IGF-1 (100 ng/ml) for 1-5 min with or without 100 nM Wortmannin. Cell extracts (500 μg protein) were immunoprecipitated with anti-Akt antibodies and kinase activity was determined by using an Akt/PKB assay kit from Upstate Biotechnology. The basal Akt activity was ~4.8 pmol 32 Pi incorporated into Akt peptides/0.5 mg protein/10 min (n=5). (B) Mesangial cells were serum-starved overnight and incubated with IGF-1 (100 ng/ml) for different time periods: 0, 1, 3, 5, and 10 min, respectively, and 30 μg proteins were subjected to Western Blotting with anti-phospho-GSK-3β antibodies (n=3). The membrane was stripped and reprobed with anti-GSK-3α/β antibodies. (C) Mesangial cells were treated with IGF-1 (100 ng/ml) for 0, 2, 6 and 12 h and were subjected to Western blotting for cyclin D1 and reprobed for α-tubulin (a representative of n=3).
    Figure Legend Snippet: Effect of IGF-1 on AKT/GSK-3β activity and cyclin D1 expression in mesangial cells. (A) Mesangial cells were grown to 60-70% confluence, serum-starved overnight and treated with IGF-1 (100 ng/ml) for 1-5 min with or without 100 nM Wortmannin. Cell extracts (500 μg protein) were immunoprecipitated with anti-Akt antibodies and kinase activity was determined by using an Akt/PKB assay kit from Upstate Biotechnology. The basal Akt activity was ~4.8 pmol 32 Pi incorporated into Akt peptides/0.5 mg protein/10 min (n=5). (B) Mesangial cells were serum-starved overnight and incubated with IGF-1 (100 ng/ml) for different time periods: 0, 1, 3, 5, and 10 min, respectively, and 30 μg proteins were subjected to Western Blotting with anti-phospho-GSK-3β antibodies (n=3). The membrane was stripped and reprobed with anti-GSK-3α/β antibodies. (C) Mesangial cells were treated with IGF-1 (100 ng/ml) for 0, 2, 6 and 12 h and were subjected to Western blotting for cyclin D1 and reprobed for α-tubulin (a representative of n=3).

    Techniques Used: Activity Assay, Expressing, Immunoprecipitation, Incubation, Western Blot

    Effect of Akt and GSK-3β inhibition on CREB phosphorylation and cyclin D1 and fibronectin expression. Mesangial cells were grown to 70% confluence and serum-starved overnight. Inhibitors for Akt/PKB (2 μM, SH-5) and GSK-β (100 nM, SB216763) were pre-treated for 30 min before adding IGF-1 (100 ng/ml) for 2 h. Cell extracts (30 μg protein) were subjected to Western blotting for (A) phosphorylated and total CREB and (B) for cyclin D1. Lane 1. Control; lane 2. IGF-1, 2h; lane 3. IGF-1, 2h+SH-5 (2 μM); and lane 4. IGF-1, 2h+SB216763 (100 nM). Representative blots from three different experiments are shown. (C). Effect of IGF-1 (100 ng/ml) or SB216763 (100 nM) on fibronectin expression upon 24 h exposure was determined by immunofluorescence microscopy. Nuclear staining was performed with Hoechst Dye 33342 and fibronectin with Alexis 488 (n=3).
    Figure Legend Snippet: Effect of Akt and GSK-3β inhibition on CREB phosphorylation and cyclin D1 and fibronectin expression. Mesangial cells were grown to 70% confluence and serum-starved overnight. Inhibitors for Akt/PKB (2 μM, SH-5) and GSK-β (100 nM, SB216763) were pre-treated for 30 min before adding IGF-1 (100 ng/ml) for 2 h. Cell extracts (30 μg protein) were subjected to Western blotting for (A) phosphorylated and total CREB and (B) for cyclin D1. Lane 1. Control; lane 2. IGF-1, 2h; lane 3. IGF-1, 2h+SH-5 (2 μM); and lane 4. IGF-1, 2h+SB216763 (100 nM). Representative blots from three different experiments are shown. (C). Effect of IGF-1 (100 ng/ml) or SB216763 (100 nM) on fibronectin expression upon 24 h exposure was determined by immunofluorescence microscopy. Nuclear staining was performed with Hoechst Dye 33342 and fibronectin with Alexis 488 (n=3).

    Techniques Used: Inhibition, Expressing, Western Blot, Immunofluorescence, Microscopy, Staining

    IGF-1 activation of Akt substrate phosphorylation and interaction of phospho-GSK-3β with 14-3-3ζ. Mesangial cells were grown to 70-80% confluence and serum-starved overnight. IGF-1 (100 ng/ml) was added for 2 h and cells were harvested as described in Methods. Total extracts were subjected to SDS-PAGE and Western blotting with phospho-Akt motif antibodies (Figure A, left panel). 14-3-3ζ immunoprecipitates were probed with the phospho-Akt motif antibodies (Figure A, right panel). In addition, the 14-3-3 ζ immunoprecipitates were subjected to Western blotting for 14-3-3 ζ, GSK-3α/β, phosphorylated GSK-3β(S9P) and β-catenin (Figure B). The blots are representatives of three repeats.
    Figure Legend Snippet: IGF-1 activation of Akt substrate phosphorylation and interaction of phospho-GSK-3β with 14-3-3ζ. Mesangial cells were grown to 70-80% confluence and serum-starved overnight. IGF-1 (100 ng/ml) was added for 2 h and cells were harvested as described in Methods. Total extracts were subjected to SDS-PAGE and Western blotting with phospho-Akt motif antibodies (Figure A, left panel). 14-3-3ζ immunoprecipitates were probed with the phospho-Akt motif antibodies (Figure A, right panel). In addition, the 14-3-3 ζ immunoprecipitates were subjected to Western blotting for 14-3-3 ζ, GSK-3α/β, phosphorylated GSK-3β(S9P) and β-catenin (Figure B). The blots are representatives of three repeats.

    Techniques Used: Activation Assay, SDS Page, Western Blot

    anti glcnac s t antibody beads  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti glcnac s t antibody beads
    Anti Glcnac S T Antibody Beads, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti akt substrate rxxps t antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti akt substrate rxxps t antibody
    Anti Akt Substrate Rxxps T Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho akt substrate rxxs t  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho akt substrate rxxs t
    Phospho Akt Substrate Rxxs T, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti phospho pkc substrate motif r k  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti phospho pkc substrate motif r k
    Anti Phospho Pkc Substrate Motif R K, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho akt substrate rxxs t  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho akt substrate rxxs t
    Phospho Akt Substrate Rxxs T, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    phospho pkc substrate motif  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho pkc substrate motif
    Phorbol 12-Myristate 13-Acetate (PMA) treatment induces phospho-protein kinase C <t>(PKC)</t> activation and MO3.13 differentiation. ( A ) Western blot analysis showing the activation rate of PKC substrates in MO3.13 cells after 100 nM PMA short-term treatment (5 min and 15 min). An anti-phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody was used. This antibody specifically detects endogenous levels of cellular proteins only when phosphorylated at Ser residues (S) surrounded by Arg (R) or Lys (K) at the ⁻2 and ⁺2 positions. p38 was used as a loading control. ( B ) Western blot analysis exhibiting the activation degree of PKC substrates after 96 h of PMA treatment compared to the control (CTR). p38 was used as a loading control. ( C ) Western blot analysis reporting the activation rate of AKT substrates, MARCKS and ERK proteins at 1 h of PMA treatment. ( D ) Western blot analysis reporting the activation of PKC substrates in MO3.13 cells, after treatment with PMA and Ro 31-8220 (1 μM) used alone and in combination for 1 h. p38 was used as a loading control. ( E ) Representative images of MO3.13 cells cultured in a normal condition medium (left) or treated with 100 nM PMA (right) for 96 h. Images were acquired using an inverted wide-field microscope (Olympus IX51). Scale bar 200 μm. ( F ) MO3.13 cell proliferation assessed by MTT test for 24 h, 48 h, 72 h and 96 h. Data were represented as the number of viable cells compared to the CTR, expressed as mean ± standard deviation (SD). **** p -value < 0.0001 by t -test. ( G ) Western blot analysis was performed on the whole-cell lysate using an anti-p27 antibody in MO3.13 cells treated with PMA for 48 h and 96 h. p38 was used as a loading control. ( H ) Western blot analyses using anti-β-tubulin3, anti-MBP, anti-GFAP, anti-E-cadherin, anti-Myc and <t>anti-β-catenin</t> <t>antibodies</t> were performed on the whole protein extract of MO3.13, CTR and treated conditions. p38 was used as a loading control. Western blot experiments were performed in triplicates.
    Phospho Pkc Substrate Motif, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks"

    Article Title: Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22105245

    Phorbol 12-Myristate 13-Acetate (PMA) treatment induces phospho-protein kinase C (PKC) activation and MO3.13 differentiation. ( A ) Western blot analysis showing the activation rate of PKC substrates in MO3.13 cells after 100 nM PMA short-term treatment (5 min and 15 min). An anti-phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody was used. This antibody specifically detects endogenous levels of cellular proteins only when phosphorylated at Ser residues (S) surrounded by Arg (R) or Lys (K) at the ⁻2 and ⁺2 positions. p38 was used as a loading control. ( B ) Western blot analysis exhibiting the activation degree of PKC substrates after 96 h of PMA treatment compared to the control (CTR). p38 was used as a loading control. ( C ) Western blot analysis reporting the activation rate of AKT substrates, MARCKS and ERK proteins at 1 h of PMA treatment. ( D ) Western blot analysis reporting the activation of PKC substrates in MO3.13 cells, after treatment with PMA and Ro 31-8220 (1 μM) used alone and in combination for 1 h. p38 was used as a loading control. ( E ) Representative images of MO3.13 cells cultured in a normal condition medium (left) or treated with 100 nM PMA (right) for 96 h. Images were acquired using an inverted wide-field microscope (Olympus IX51). Scale bar 200 μm. ( F ) MO3.13 cell proliferation assessed by MTT test for 24 h, 48 h, 72 h and 96 h. Data were represented as the number of viable cells compared to the CTR, expressed as mean ± standard deviation (SD). **** p -value < 0.0001 by t -test. ( G ) Western blot analysis was performed on the whole-cell lysate using an anti-p27 antibody in MO3.13 cells treated with PMA for 48 h and 96 h. p38 was used as a loading control. ( H ) Western blot analyses using anti-β-tubulin3, anti-MBP, anti-GFAP, anti-E-cadherin, anti-Myc and anti-β-catenin antibodies were performed on the whole protein extract of MO3.13, CTR and treated conditions. p38 was used as a loading control. Western blot experiments were performed in triplicates.
    Figure Legend Snippet: Phorbol 12-Myristate 13-Acetate (PMA) treatment induces phospho-protein kinase C (PKC) activation and MO3.13 differentiation. ( A ) Western blot analysis showing the activation rate of PKC substrates in MO3.13 cells after 100 nM PMA short-term treatment (5 min and 15 min). An anti-phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody was used. This antibody specifically detects endogenous levels of cellular proteins only when phosphorylated at Ser residues (S) surrounded by Arg (R) or Lys (K) at the ⁻2 and ⁺2 positions. p38 was used as a loading control. ( B ) Western blot analysis exhibiting the activation degree of PKC substrates after 96 h of PMA treatment compared to the control (CTR). p38 was used as a loading control. ( C ) Western blot analysis reporting the activation rate of AKT substrates, MARCKS and ERK proteins at 1 h of PMA treatment. ( D ) Western blot analysis reporting the activation of PKC substrates in MO3.13 cells, after treatment with PMA and Ro 31-8220 (1 μM) used alone and in combination for 1 h. p38 was used as a loading control. ( E ) Representative images of MO3.13 cells cultured in a normal condition medium (left) or treated with 100 nM PMA (right) for 96 h. Images were acquired using an inverted wide-field microscope (Olympus IX51). Scale bar 200 μm. ( F ) MO3.13 cell proliferation assessed by MTT test for 24 h, 48 h, 72 h and 96 h. Data were represented as the number of viable cells compared to the CTR, expressed as mean ± standard deviation (SD). **** p -value < 0.0001 by t -test. ( G ) Western blot analysis was performed on the whole-cell lysate using an anti-p27 antibody in MO3.13 cells treated with PMA for 48 h and 96 h. p38 was used as a loading control. ( H ) Western blot analyses using anti-β-tubulin3, anti-MBP, anti-GFAP, anti-E-cadherin, anti-Myc and anti-β-catenin antibodies were performed on the whole protein extract of MO3.13, CTR and treated conditions. p38 was used as a loading control. Western blot experiments were performed in triplicates.

    Techniques Used: Activation Assay, Western Blot, Cell Culture, Microscopy, Standard Deviation

    Signaling pathways and transcriptional factors modulated by PKC activation. ( A ) “Regulation of actin cytoskeleton” KEGG pathway is visualized. Red stars indicated differentially expressed proteins involved in the pathway and identified by MS/MS analysis, or Western blotting, after PMA treatment. ( B ) Expression profile of cofilin and phospho-cofilin was detected by Western blot at 0 h, 24 h, 48 h, 72 h and 96 h after treatment with PMA. Histogram represents the expression ratio of phospho-cofilin of the treated condition versus CTR sample. *** p -value < 0.001. ( C ) Phase-contrast images of MO3.13 cells cultured in a normal condition medium or treated with 100 nM PMA and 10 μM Y-27632, alone or in combination, for 96 h. Scale bar 200 μm. ( D ) Western blot analysis showing the activation level of PKC substrates in control and treated conditions after 1 h of stimulation. PMA at 100 nM and Y-27632 at 10 μM were used alone or in combination. The detection was performed through a phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody. The Ponceau S stained membrane is shown to verify the protein loading. ( E ) Western blot analysis showing the expression levels of cofilin and phospho-cofilin after treatment with PMA and Y-27632, used alone or in combination, for 96 h. Histogram represents the expression ratio of phospho-cofilin in the treated conditions versus the CTR sample. *** p -value < 0.001. ( F ) Hierarchical clustering based on Euclidean distance showing the separation between the CTR cells and those treated with PMA and Y-27632, alone and in combination. Profile plots of selected protein groups are shown, displaying distinct behavior concerning treatment.
    Figure Legend Snippet: Signaling pathways and transcriptional factors modulated by PKC activation. ( A ) “Regulation of actin cytoskeleton” KEGG pathway is visualized. Red stars indicated differentially expressed proteins involved in the pathway and identified by MS/MS analysis, or Western blotting, after PMA treatment. ( B ) Expression profile of cofilin and phospho-cofilin was detected by Western blot at 0 h, 24 h, 48 h, 72 h and 96 h after treatment with PMA. Histogram represents the expression ratio of phospho-cofilin of the treated condition versus CTR sample. *** p -value < 0.001. ( C ) Phase-contrast images of MO3.13 cells cultured in a normal condition medium or treated with 100 nM PMA and 10 μM Y-27632, alone or in combination, for 96 h. Scale bar 200 μm. ( D ) Western blot analysis showing the activation level of PKC substrates in control and treated conditions after 1 h of stimulation. PMA at 100 nM and Y-27632 at 10 μM were used alone or in combination. The detection was performed through a phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody. The Ponceau S stained membrane is shown to verify the protein loading. ( E ) Western blot analysis showing the expression levels of cofilin and phospho-cofilin after treatment with PMA and Y-27632, used alone or in combination, for 96 h. Histogram represents the expression ratio of phospho-cofilin in the treated conditions versus the CTR sample. *** p -value < 0.001. ( F ) Hierarchical clustering based on Euclidean distance showing the separation between the CTR cells and those treated with PMA and Y-27632, alone and in combination. Profile plots of selected protein groups are shown, displaying distinct behavior concerning treatment.

    Techniques Used: Activation Assay, Tandem Mass Spectroscopy, Western Blot, Expressing, Cell Culture, Staining

    phospho pkc substrate motif  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc phospho pkc substrate motif
    mTOR regulates CRP-induced phosphorylation of PKCs. Platelets from WT (W) or mTOR −/− (KO, K) mice were stimulated with CRP at the indicated low concentration for 5.5 min. Lysates of platelets were immunoblotted <t>with</t> <t>antibodies</t> against a , b phospho-PKCα Thr497 and PKCα, c , d phospho-PKCβ Thr641 and PKCβ, c , e phospho-PKCβ Ser660 and <t>PKC</t> β, f , g phospho-PKCθ Thr538 and PKCθ, h , i phospho-PKCδ Thr505 and PKCδ, and j , k phospho-PKCε Ser729 and PKCε; these images (separated by horizontal white space) were cropped from the different/same gels and full-length/original blots were shown in Additional file : Part III. Phosphoprotein levels were normalized to total protein levels and were standardized to 1 in unstimulated WT samples. Values represent means ± SEM from at least three independent experiments (*P < 0.05, **P < 0.01; paired Student’s t test)
    Phospho Pkc Substrate Motif, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "mTOR regulates GPVI-mediated platelet activation"

    Article Title: mTOR regulates GPVI-mediated platelet activation

    Journal: Journal of Translational Medicine

    doi: 10.1186/s12967-021-02756-y

    mTOR regulates CRP-induced phosphorylation of PKCs. Platelets from WT (W) or mTOR −/− (KO, K) mice were stimulated with CRP at the indicated low concentration for 5.5 min. Lysates of platelets were immunoblotted with antibodies against a , b phospho-PKCα Thr497 and PKCα, c , d phospho-PKCβ Thr641 and PKCβ, c , e phospho-PKCβ Ser660 and PKC β, f , g phospho-PKCθ Thr538 and PKCθ, h , i phospho-PKCδ Thr505 and PKCδ, and j , k phospho-PKCε Ser729 and PKCε; these images (separated by horizontal white space) were cropped from the different/same gels and full-length/original blots were shown in Additional file : Part III. Phosphoprotein levels were normalized to total protein levels and were standardized to 1 in unstimulated WT samples. Values represent means ± SEM from at least three independent experiments (*P < 0.05, **P < 0.01; paired Student’s t test)
    Figure Legend Snippet: mTOR regulates CRP-induced phosphorylation of PKCs. Platelets from WT (W) or mTOR −/− (KO, K) mice were stimulated with CRP at the indicated low concentration for 5.5 min. Lysates of platelets were immunoblotted with antibodies against a , b phospho-PKCα Thr497 and PKCα, c , d phospho-PKCβ Thr641 and PKCβ, c , e phospho-PKCβ Ser660 and PKC β, f , g phospho-PKCθ Thr538 and PKCθ, h , i phospho-PKCδ Thr505 and PKCδ, and j , k phospho-PKCε Ser729 and PKCε; these images (separated by horizontal white space) were cropped from the different/same gels and full-length/original blots were shown in Additional file : Part III. Phosphoprotein levels were normalized to total protein levels and were standardized to 1 in unstimulated WT samples. Values represent means ± SEM from at least three independent experiments (*P < 0.05, **P < 0.01; paired Student’s t test)

    Techniques Used: Concentration Assay

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    Cell Signaling Technology Inc phospho pkc substrate motif
    Phospho Pkc Substrate Motif, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho rxx s t antibody
    hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif <t>RXX(s/t).</t> b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.
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    Phorbol 12-Myristate 13-Acetate (PMA) treatment induces phospho-protein kinase C <t>(PKC)</t> activation and MO3.13 differentiation. ( A ) Western blot analysis showing the activation rate of PKC substrates in MO3.13 cells after 100 nM PMA short-term treatment (5 min and 15 min). An anti-phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody was used. This antibody specifically detects endogenous levels of cellular proteins only when phosphorylated at Ser residues (S) surrounded by Arg (R) or Lys (K) at the ⁻2 and ⁺2 positions. p38 was used as a loading control. ( B ) Western blot analysis exhibiting the activation degree of PKC substrates after 96 h of PMA treatment compared to the control (CTR). p38 was used as a loading control. ( C ) Western blot analysis reporting the activation rate of AKT substrates, MARCKS and ERK proteins at 1 h of PMA treatment. ( D ) Western blot analysis reporting the activation of PKC substrates in MO3.13 cells, after treatment with PMA and Ro 31-8220 (1 μM) used alone and in combination for 1 h. p38 was used as a loading control. ( E ) Representative images of MO3.13 cells cultured in a normal condition medium (left) or treated with 100 nM PMA (right) for 96 h. Images were acquired using an inverted wide-field microscope (Olympus IX51). Scale bar 200 μm. ( F ) MO3.13 cell proliferation assessed by MTT test for 24 h, 48 h, 72 h and 96 h. Data were represented as the number of viable cells compared to the CTR, expressed as mean ± standard deviation (SD). **** p -value < 0.0001 by t -test. ( G ) Western blot analysis was performed on the whole-cell lysate using an anti-p27 antibody in MO3.13 cells treated with PMA for 48 h and 96 h. p38 was used as a loading control. ( H ) Western blot analyses using anti-β-tubulin3, anti-MBP, anti-GFAP, anti-E-cadherin, anti-Myc and <t>anti-β-catenin</t> <t>antibodies</t> were performed on the whole protein extract of MO3.13, CTR and treated conditions. p38 was used as a loading control. Western blot experiments were performed in triplicates.
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    hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif RXX(s/t). b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.

    Journal: PLoS ONE

    Article Title: Fibroblast Growth Factor 21 Improves Insulin Sensitivity and Synergizes with Insulin in Human Adipose Stem Cell-Derived (hASC) Adipocytes

    doi: 10.1371/journal.pone.0111767

    Figure Lengend Snippet: hASC-adipocytes were treated with FGF21 (10 nM) either alone or in combination with insulin (10 nM) for 30 min prior to harvest. hASC-adipocytes. a ) Levels of phosphorylation of Akt substrates were assessed by western blot using a phospho-specific antibody recognizing proteins with the motif RXX(s/t). b ) Levels of phosphorylation of ATM/ATR substrates were determined using a phospho-specific antibody recognizing proteins with the motif sQ.

    Article Snippet: 10 mg total protein was immunoaffinity-purified with anti-phospho-RXX(s/t) antibody for Akt substrates (CST 9614) or anti-phospho-(s)Q antibody for ATM/ATR substrates.

    Techniques: Western Blot

    Effect of IGF-1 on AKT/GSK-3β activity and cyclin D1 expression in mesangial cells. (A) Mesangial cells were grown to 60-70% confluence, serum-starved overnight and treated with IGF-1 (100 ng/ml) for 1-5 min with or without 100 nM Wortmannin. Cell extracts (500 μg protein) were immunoprecipitated with anti-Akt antibodies and kinase activity was determined by using an Akt/PKB assay kit from Upstate Biotechnology. The basal Akt activity was ~4.8 pmol 32 Pi incorporated into Akt peptides/0.5 mg protein/10 min (n=5). (B) Mesangial cells were serum-starved overnight and incubated with IGF-1 (100 ng/ml) for different time periods: 0, 1, 3, 5, and 10 min, respectively, and 30 μg proteins were subjected to Western Blotting with anti-phospho-GSK-3β antibodies (n=3). The membrane was stripped and reprobed with anti-GSK-3α/β antibodies. (C) Mesangial cells were treated with IGF-1 (100 ng/ml) for 0, 2, 6 and 12 h and were subjected to Western blotting for cyclin D1 and reprobed for α-tubulin (a representative of n=3).

    Journal: International Journal of Biological Sciences

    Article Title: Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

    doi:

    Figure Lengend Snippet: Effect of IGF-1 on AKT/GSK-3β activity and cyclin D1 expression in mesangial cells. (A) Mesangial cells were grown to 60-70% confluence, serum-starved overnight and treated with IGF-1 (100 ng/ml) for 1-5 min with or without 100 nM Wortmannin. Cell extracts (500 μg protein) were immunoprecipitated with anti-Akt antibodies and kinase activity was determined by using an Akt/PKB assay kit from Upstate Biotechnology. The basal Akt activity was ~4.8 pmol 32 Pi incorporated into Akt peptides/0.5 mg protein/10 min (n=5). (B) Mesangial cells were serum-starved overnight and incubated with IGF-1 (100 ng/ml) for different time periods: 0, 1, 3, 5, and 10 min, respectively, and 30 μg proteins were subjected to Western Blotting with anti-phospho-GSK-3β antibodies (n=3). The membrane was stripped and reprobed with anti-GSK-3α/β antibodies. (C) Mesangial cells were treated with IGF-1 (100 ng/ml) for 0, 2, 6 and 12 h and were subjected to Western blotting for cyclin D1 and reprobed for α-tubulin (a representative of n=3).

    Article Snippet: Anti-cyclin D1 and phosphorylated Akt substrate motif specific antibodies were purchased form Cell Signaling Technology, Danvers, MA.

    Techniques: Activity Assay, Expressing, Immunoprecipitation, Incubation, Western Blot

    Effect of Akt and GSK-3β inhibition on CREB phosphorylation and cyclin D1 and fibronectin expression. Mesangial cells were grown to 70% confluence and serum-starved overnight. Inhibitors for Akt/PKB (2 μM, SH-5) and GSK-β (100 nM, SB216763) were pre-treated for 30 min before adding IGF-1 (100 ng/ml) for 2 h. Cell extracts (30 μg protein) were subjected to Western blotting for (A) phosphorylated and total CREB and (B) for cyclin D1. Lane 1. Control; lane 2. IGF-1, 2h; lane 3. IGF-1, 2h+SH-5 (2 μM); and lane 4. IGF-1, 2h+SB216763 (100 nM). Representative blots from three different experiments are shown. (C). Effect of IGF-1 (100 ng/ml) or SB216763 (100 nM) on fibronectin expression upon 24 h exposure was determined by immunofluorescence microscopy. Nuclear staining was performed with Hoechst Dye 33342 and fibronectin with Alexis 488 (n=3).

    Journal: International Journal of Biological Sciences

    Article Title: Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

    doi:

    Figure Lengend Snippet: Effect of Akt and GSK-3β inhibition on CREB phosphorylation and cyclin D1 and fibronectin expression. Mesangial cells were grown to 70% confluence and serum-starved overnight. Inhibitors for Akt/PKB (2 μM, SH-5) and GSK-β (100 nM, SB216763) were pre-treated for 30 min before adding IGF-1 (100 ng/ml) for 2 h. Cell extracts (30 μg protein) were subjected to Western blotting for (A) phosphorylated and total CREB and (B) for cyclin D1. Lane 1. Control; lane 2. IGF-1, 2h; lane 3. IGF-1, 2h+SH-5 (2 μM); and lane 4. IGF-1, 2h+SB216763 (100 nM). Representative blots from three different experiments are shown. (C). Effect of IGF-1 (100 ng/ml) or SB216763 (100 nM) on fibronectin expression upon 24 h exposure was determined by immunofluorescence microscopy. Nuclear staining was performed with Hoechst Dye 33342 and fibronectin with Alexis 488 (n=3).

    Article Snippet: Anti-cyclin D1 and phosphorylated Akt substrate motif specific antibodies were purchased form Cell Signaling Technology, Danvers, MA.

    Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence, Microscopy, Staining

    IGF-1 activation of Akt substrate phosphorylation and interaction of phospho-GSK-3β with 14-3-3ζ. Mesangial cells were grown to 70-80% confluence and serum-starved overnight. IGF-1 (100 ng/ml) was added for 2 h and cells were harvested as described in Methods. Total extracts were subjected to SDS-PAGE and Western blotting with phospho-Akt motif antibodies (Figure A, left panel). 14-3-3ζ immunoprecipitates were probed with the phospho-Akt motif antibodies (Figure A, right panel). In addition, the 14-3-3 ζ immunoprecipitates were subjected to Western blotting for 14-3-3 ζ, GSK-3α/β, phosphorylated GSK-3β(S9P) and β-catenin (Figure B). The blots are representatives of three repeats.

    Journal: International Journal of Biological Sciences

    Article Title: Proteomic Identification of 14-3-3ζ as an Adapter for IGF-1 and Akt/GSK-3β Signaling and Survival of Renal Mesangial Cells

    doi:

    Figure Lengend Snippet: IGF-1 activation of Akt substrate phosphorylation and interaction of phospho-GSK-3β with 14-3-3ζ. Mesangial cells were grown to 70-80% confluence and serum-starved overnight. IGF-1 (100 ng/ml) was added for 2 h and cells were harvested as described in Methods. Total extracts were subjected to SDS-PAGE and Western blotting with phospho-Akt motif antibodies (Figure A, left panel). 14-3-3ζ immunoprecipitates were probed with the phospho-Akt motif antibodies (Figure A, right panel). In addition, the 14-3-3 ζ immunoprecipitates were subjected to Western blotting for 14-3-3 ζ, GSK-3α/β, phosphorylated GSK-3β(S9P) and β-catenin (Figure B). The blots are representatives of three repeats.

    Article Snippet: Anti-cyclin D1 and phosphorylated Akt substrate motif specific antibodies were purchased form Cell Signaling Technology, Danvers, MA.

    Techniques: Activation Assay, SDS Page, Western Blot

    Phorbol 12-Myristate 13-Acetate (PMA) treatment induces phospho-protein kinase C (PKC) activation and MO3.13 differentiation. ( A ) Western blot analysis showing the activation rate of PKC substrates in MO3.13 cells after 100 nM PMA short-term treatment (5 min and 15 min). An anti-phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody was used. This antibody specifically detects endogenous levels of cellular proteins only when phosphorylated at Ser residues (S) surrounded by Arg (R) or Lys (K) at the ⁻2 and ⁺2 positions. p38 was used as a loading control. ( B ) Western blot analysis exhibiting the activation degree of PKC substrates after 96 h of PMA treatment compared to the control (CTR). p38 was used as a loading control. ( C ) Western blot analysis reporting the activation rate of AKT substrates, MARCKS and ERK proteins at 1 h of PMA treatment. ( D ) Western blot analysis reporting the activation of PKC substrates in MO3.13 cells, after treatment with PMA and Ro 31-8220 (1 μM) used alone and in combination for 1 h. p38 was used as a loading control. ( E ) Representative images of MO3.13 cells cultured in a normal condition medium (left) or treated with 100 nM PMA (right) for 96 h. Images were acquired using an inverted wide-field microscope (Olympus IX51). Scale bar 200 μm. ( F ) MO3.13 cell proliferation assessed by MTT test for 24 h, 48 h, 72 h and 96 h. Data were represented as the number of viable cells compared to the CTR, expressed as mean ± standard deviation (SD). **** p -value < 0.0001 by t -test. ( G ) Western blot analysis was performed on the whole-cell lysate using an anti-p27 antibody in MO3.13 cells treated with PMA for 48 h and 96 h. p38 was used as a loading control. ( H ) Western blot analyses using anti-β-tubulin3, anti-MBP, anti-GFAP, anti-E-cadherin, anti-Myc and anti-β-catenin antibodies were performed on the whole protein extract of MO3.13, CTR and treated conditions. p38 was used as a loading control. Western blot experiments were performed in triplicates.

    Journal: International Journal of Molecular Sciences

    Article Title: Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks

    doi: 10.3390/ijms22105245

    Figure Lengend Snippet: Phorbol 12-Myristate 13-Acetate (PMA) treatment induces phospho-protein kinase C (PKC) activation and MO3.13 differentiation. ( A ) Western blot analysis showing the activation rate of PKC substrates in MO3.13 cells after 100 nM PMA short-term treatment (5 min and 15 min). An anti-phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody was used. This antibody specifically detects endogenous levels of cellular proteins only when phosphorylated at Ser residues (S) surrounded by Arg (R) or Lys (K) at the ⁻2 and ⁺2 positions. p38 was used as a loading control. ( B ) Western blot analysis exhibiting the activation degree of PKC substrates after 96 h of PMA treatment compared to the control (CTR). p38 was used as a loading control. ( C ) Western blot analysis reporting the activation rate of AKT substrates, MARCKS and ERK proteins at 1 h of PMA treatment. ( D ) Western blot analysis reporting the activation of PKC substrates in MO3.13 cells, after treatment with PMA and Ro 31-8220 (1 μM) used alone and in combination for 1 h. p38 was used as a loading control. ( E ) Representative images of MO3.13 cells cultured in a normal condition medium (left) or treated with 100 nM PMA (right) for 96 h. Images were acquired using an inverted wide-field microscope (Olympus IX51). Scale bar 200 μm. ( F ) MO3.13 cell proliferation assessed by MTT test for 24 h, 48 h, 72 h and 96 h. Data were represented as the number of viable cells compared to the CTR, expressed as mean ± standard deviation (SD). **** p -value < 0.0001 by t -test. ( G ) Western blot analysis was performed on the whole-cell lysate using an anti-p27 antibody in MO3.13 cells treated with PMA for 48 h and 96 h. p38 was used as a loading control. ( H ) Western blot analyses using anti-β-tubulin3, anti-MBP, anti-GFAP, anti-E-cadherin, anti-Myc and anti-β-catenin antibodies were performed on the whole protein extract of MO3.13, CTR and treated conditions. p38 was used as a loading control. Western blot experiments were performed in triplicates.

    Article Snippet: Membranes were probed with the following antibodies (1:1000 dilution): phospho-PKC substrate motif ((R/K)XPSX(R/K)) MultiMabTM (#6967), MARCKS (#5607), Phospho-MARCKS (Ser167/170) (8722), p38 (#9212), c-Myc (#13987), myelin basic protein (#78896), β-catenin (#8480), phospho-ERK1/2 (#4370), ERK1/2 (#4695), phospho-AKT substrate (RXXS*/T*) (#9614S), PKCα (#9960), SQSTM1/p62 (#39749) were from Cell Signaling.

    Techniques: Activation Assay, Western Blot, Cell Culture, Microscopy, Standard Deviation

    Signaling pathways and transcriptional factors modulated by PKC activation. ( A ) “Regulation of actin cytoskeleton” KEGG pathway is visualized. Red stars indicated differentially expressed proteins involved in the pathway and identified by MS/MS analysis, or Western blotting, after PMA treatment. ( B ) Expression profile of cofilin and phospho-cofilin was detected by Western blot at 0 h, 24 h, 48 h, 72 h and 96 h after treatment with PMA. Histogram represents the expression ratio of phospho-cofilin of the treated condition versus CTR sample. *** p -value < 0.001. ( C ) Phase-contrast images of MO3.13 cells cultured in a normal condition medium or treated with 100 nM PMA and 10 μM Y-27632, alone or in combination, for 96 h. Scale bar 200 μm. ( D ) Western blot analysis showing the activation level of PKC substrates in control and treated conditions after 1 h of stimulation. PMA at 100 nM and Y-27632 at 10 μM were used alone or in combination. The detection was performed through a phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody. The Ponceau S stained membrane is shown to verify the protein loading. ( E ) Western blot analysis showing the expression levels of cofilin and phospho-cofilin after treatment with PMA and Y-27632, used alone or in combination, for 96 h. Histogram represents the expression ratio of phospho-cofilin in the treated conditions versus the CTR sample. *** p -value < 0.001. ( F ) Hierarchical clustering based on Euclidean distance showing the separation between the CTR cells and those treated with PMA and Y-27632, alone and in combination. Profile plots of selected protein groups are shown, displaying distinct behavior concerning treatment.

    Journal: International Journal of Molecular Sciences

    Article Title: Protein Kinase C Activation Drives a Differentiation Program in an Oligodendroglial Precursor Model through the Modulation of Specific Biological Networks

    doi: 10.3390/ijms22105245

    Figure Lengend Snippet: Signaling pathways and transcriptional factors modulated by PKC activation. ( A ) “Regulation of actin cytoskeleton” KEGG pathway is visualized. Red stars indicated differentially expressed proteins involved in the pathway and identified by MS/MS analysis, or Western blotting, after PMA treatment. ( B ) Expression profile of cofilin and phospho-cofilin was detected by Western blot at 0 h, 24 h, 48 h, 72 h and 96 h after treatment with PMA. Histogram represents the expression ratio of phospho-cofilin of the treated condition versus CTR sample. *** p -value < 0.001. ( C ) Phase-contrast images of MO3.13 cells cultured in a normal condition medium or treated with 100 nM PMA and 10 μM Y-27632, alone or in combination, for 96 h. Scale bar 200 μm. ( D ) Western blot analysis showing the activation level of PKC substrates in control and treated conditions after 1 h of stimulation. PMA at 100 nM and Y-27632 at 10 μM were used alone or in combination. The detection was performed through a phospho-PKC substrate motif ((R/K)XpSX(R/K)) antibody. The Ponceau S stained membrane is shown to verify the protein loading. ( E ) Western blot analysis showing the expression levels of cofilin and phospho-cofilin after treatment with PMA and Y-27632, used alone or in combination, for 96 h. Histogram represents the expression ratio of phospho-cofilin in the treated conditions versus the CTR sample. *** p -value < 0.001. ( F ) Hierarchical clustering based on Euclidean distance showing the separation between the CTR cells and those treated with PMA and Y-27632, alone and in combination. Profile plots of selected protein groups are shown, displaying distinct behavior concerning treatment.

    Article Snippet: Membranes were probed with the following antibodies (1:1000 dilution): phospho-PKC substrate motif ((R/K)XPSX(R/K)) MultiMabTM (#6967), MARCKS (#5607), Phospho-MARCKS (Ser167/170) (8722), p38 (#9212), c-Myc (#13987), myelin basic protein (#78896), β-catenin (#8480), phospho-ERK1/2 (#4370), ERK1/2 (#4695), phospho-AKT substrate (RXXS*/T*) (#9614S), PKCα (#9960), SQSTM1/p62 (#39749) were from Cell Signaling.

    Techniques: Activation Assay, Tandem Mass Spectroscopy, Western Blot, Expressing, Cell Culture, Staining