2 mercaptoethanol me  (Thermo Fisher)


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    Structured Review

    Thermo Fisher 2 mercaptoethanol me
    2 Mercaptoethanol Me, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    Article Title: In vitro assessment of the feline cell-mediated immune response against feline panleukopeniavirus, calicivirus and felid herpesvirus 1 using 5-bromo-2′-deoxyuridine labeling
    Article Snippet: .. After antigen pulsing, cells were washed and further incubated with 5 × 105 ml−1 homologous non-adherent cells stored in liquid nitrogen (N2 ) for 4 days at 37 °C, 5% CO2 in complete RPMI 1640 medium containing 5% FBS (Invitrogen 16000 ) instead of 10% and 0.05 mM 2-mercaptoethanol (ME) (Invitrogen). .. Cells were then stained for proliferation with the FITC BrdU flow kit according to manufacturer's instructions (Becton, Dickinson and Company, NJ, USA).

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    Thermo Fisher stat1
    Schematic representation of the putative interaction of <t>STAT1</t> with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.
    Stat1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher deta
    Co-culture of OPCs with EMNs and MBP expression on a line pattern. (A) Light microscopy images of co-cultures grown on line patterns composed of <t>DETA/SiPEG</t> SAMs, for 20 days. (B) Immunocytochemical analysis of mature MBP positive oligodendrocyte interacting
    Deta, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher non targeting sirna
    PKA modulates <t>CDK18,</t> which controls AQP2 phosphorylation at S261 and its abundance. ( A ) MCD4 cells or ( B ) murine inner medullae homogenates were left untreated, treated with forskolin (Fsk) or AVP and where indicated with a membrane-permeant version (stearate-coupled) of the PKA-specific heat-stable inhibitor peptide (S-PKI). CDK18 was immunoprecipitated (IP) and detected by Western blotting. The PKA-phosphorylated CDK18 (pSub CDK18) was detected with phospho-PKA substrate antibody. As a control, precipitations were carried out with unrelated IgG. As loading controls in input samples, HSP90 was detected. The Western blots were densitometrically evaluated. Shown are representative results from n ≥ 3 per condition. ( C ) MCD4 cells were left untransfected or transfected with non-targeting (siNT) or CDK18 <t>siRNA,</t> and stimulated with forskolin (Fsk) as indicated. AQP2 protein abundance and phosphorylation of its S256 and S261 were detected by Western blotting with specific antibodies. cg: complex glycosylated, hm: high-mannose, ng: non-glycosylated AQP2. Shown are representative results from n ≥ 8 per condition. The signals were densitometrically analysed. AQP2 mRNA expression was evaluated by PCR. Significant differences are indicated, * p
    Non Targeting Sirna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 447 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Journal: The Journal of Biological Chemistry

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    doi: 10.1074/jbc.M110.179572

    Figure Lengend Snippet: Schematic representation of the putative interaction of STAT1 with the transcription initiation machinery, leading to the modulation of apoE gene expression. Our model proposes that after DNA bending, which probably takes place during monocyte differentiation, STAT1 (bound to its site located in the 5′ end of ME.2) interacts with the transcription initiation complex, leading to the activation of apoE expression. In addition, STAT1 can interact and cooperate with other transcription factors bound on the ME.2 or on the apoE promoter for the modulation of the apoE gene expression.

    Article Snippet: The analysis of the 3′ deletion mutants of ME.2 revealed that the small 19–141 fragment did not bind STAT1 ( lane 19–141 ), whereas the larger 19–298 fragment ( lane 19–298 ) as well as the full-length ME.2 ( lane 19–619 ) bound STAT1 efficiently, suggesting that the binding site of STAT1 is located in the 141/298 region of ME.2 ( C ).

    Techniques: Expressing, Activation Assay

    Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Journal: The Journal of Biological Chemistry

    Article Title: Macrophage-specific Up-regulation of Apolipoprotein E Gene Expression by STAT1 Is Achieved via Long Range Genomic Interactions *

    doi: 10.1074/jbc.M110.179572

    Figure Lengend Snippet: Transactivation of the apoE promoter in RAW 264.7 macrophages via interaction with STAT1 transcription factor acting on the multienhancer 2. Panel A and B , RAW 264.7 cells ( A ) or HepG2 cells ( B ) were transiently transfected with plasmids [−500/+73]apoE-luc or ME.2/[−500/+73]apoE-luc in the absence ( Control ) or in the presence ( +STAT1 ) of an expression vector for STAT1. In RAW 264.7 cells, the overexpression of STAT1 did not increase the apoE promoter activity ( [ − 500 /+ 73]apoE-luc ), but the activity of the apoE promoter in the presence of ME.2 ( ME.2/[ − 500/ + 73]apoE-luc ) was augmented by STAT1 overexpression. Overexpression of STAT1 in HepG2 cells did not increase the activity of apoE either in the absence or in the presence of ME.2. Panel C , D , and E , the STAT1 binding site on ME.2 is shown. DNA pulldown assays was performed using different fragments of the ME.2 (schematic illustrated in panel D ) or with wild type or mutated 167–189 ME.2 region ( oligo wt and oligo mut ) and nuclear extract obtained from RAW 264.7 cells transfected with expression vectors for STAT1. Note that the whole ME.2 sequence (19–619) as well as the ME.2 fragments 19–298, 87–619, and 165–619 bind STAT1 transcription factor; by contrast, 19–141 as well as 267–619 fragments of ME.2 do not bind STAT1 ( panel C ). These results indicate a STAT1 binding site in the region 165–267 of ME.2. No bands appear in the negative control (“no DNA”) in which specific biotinylated DNA was replaced by biotin ( panel C ). STAT1 bound to the native 167–189 ME2 region ( panel E , lane oligo wt ), and the binding was abrogated when the STAT1 binding site was mutated ( panel E , lane oligo mut ). In the positive control, lane ME2 (19–619) , the whole ME2 sequence was used; Input represents the Western blot using the nuclear extract of RAW 264.7 cells ( panel E ).

    Article Snippet: The analysis of the 3′ deletion mutants of ME.2 revealed that the small 19–141 fragment did not bind STAT1 ( lane 19–141 ), whereas the larger 19–298 fragment ( lane 19–298 ) as well as the full-length ME.2 ( lane 19–619 ) bound STAT1 efficiently, suggesting that the binding site of STAT1 is located in the 141/298 region of ME.2 ( C ).

    Techniques: Transfection, Expressing, Plasmid Preparation, Over Expression, Activity Assay, Binding Assay, Sequencing, Negative Control, Positive Control, Western Blot

    Co-culture of OPCs with EMNs and MBP expression on a line pattern. (A) Light microscopy images of co-cultures grown on line patterns composed of DETA/SiPEG SAMs, for 20 days. (B) Immunocytochemical analysis of mature MBP positive oligodendrocyte interacting

    Journal: Journal of biomaterials and tissue engineering

    Article Title: Rat Cortical Oligodendrocyte-Embryonic Motoneuron Co-Culture: An In Vitro Axon-Oligodendrocyte Interaction Model

    doi:

    Figure Lengend Snippet: Co-culture of OPCs with EMNs and MBP expression on a line pattern. (A) Light microscopy images of co-cultures grown on line patterns composed of DETA/SiPEG SAMs, for 20 days. (B) Immunocytochemical analysis of mature MBP positive oligodendrocyte interacting

    Article Snippet: The XPS characterization of DETA and SiPEG surfaces was performed utilizing a Thermo ESCALAB 220i-XL X-Ray photoelectron spectrometer equipped with an aluminium anode and a quartz monochromator.

    Techniques: Co-Culture Assay, Expressing, Light Microscopy

    PKA modulates CDK18, which controls AQP2 phosphorylation at S261 and its abundance. ( A ) MCD4 cells or ( B ) murine inner medullae homogenates were left untreated, treated with forskolin (Fsk) or AVP and where indicated with a membrane-permeant version (stearate-coupled) of the PKA-specific heat-stable inhibitor peptide (S-PKI). CDK18 was immunoprecipitated (IP) and detected by Western blotting. The PKA-phosphorylated CDK18 (pSub CDK18) was detected with phospho-PKA substrate antibody. As a control, precipitations were carried out with unrelated IgG. As loading controls in input samples, HSP90 was detected. The Western blots were densitometrically evaluated. Shown are representative results from n ≥ 3 per condition. ( C ) MCD4 cells were left untransfected or transfected with non-targeting (siNT) or CDK18 siRNA, and stimulated with forskolin (Fsk) as indicated. AQP2 protein abundance and phosphorylation of its S256 and S261 were detected by Western blotting with specific antibodies. cg: complex glycosylated, hm: high-mannose, ng: non-glycosylated AQP2. Shown are representative results from n ≥ 8 per condition. The signals were densitometrically analysed. AQP2 mRNA expression was evaluated by PCR. Significant differences are indicated, * p

    Journal: Cells

    Article Title: Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

    doi: 10.3390/cells9030673

    Figure Lengend Snippet: PKA modulates CDK18, which controls AQP2 phosphorylation at S261 and its abundance. ( A ) MCD4 cells or ( B ) murine inner medullae homogenates were left untreated, treated with forskolin (Fsk) or AVP and where indicated with a membrane-permeant version (stearate-coupled) of the PKA-specific heat-stable inhibitor peptide (S-PKI). CDK18 was immunoprecipitated (IP) and detected by Western blotting. The PKA-phosphorylated CDK18 (pSub CDK18) was detected with phospho-PKA substrate antibody. As a control, precipitations were carried out with unrelated IgG. As loading controls in input samples, HSP90 was detected. The Western blots were densitometrically evaluated. Shown are representative results from n ≥ 3 per condition. ( C ) MCD4 cells were left untransfected or transfected with non-targeting (siNT) or CDK18 siRNA, and stimulated with forskolin (Fsk) as indicated. AQP2 protein abundance and phosphorylation of its S256 and S261 were detected by Western blotting with specific antibodies. cg: complex glycosylated, hm: high-mannose, ng: non-glycosylated AQP2. Shown are representative results from n ≥ 8 per condition. The signals were densitometrically analysed. AQP2 mRNA expression was evaluated by PCR. Significant differences are indicated, * p

    Article Snippet: CDK18 siRNA (#M-040145-01-0005/18557)), STUB1 (#M-007201-02-0005) and non-targeting siRNA (siNT; NT#2) (against firefly luciferase; #D-001206-14-20) were purchased as siGENOME SMART pools (Thermo Fisher).

    Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Polymerase Chain Reaction

    CDK18 regulates STUB1 to control AQP2 ubiquitination, abundance and localisation. ( A ) MCD4 cells were transfected with control non-targeting (siNT) or CDK18 siRNA, lysed and AQP2 was immunoprecipitated (IP). As a control, unrelated IgG was used (IgG). AQP2 and co-immunoprecipitated ubiquitin (Ubi) were detected by Western blotting (WB). Shown are a representative result from n = 4 independent experiments including the input samples where HSP90 was used as a loading control. The signals were semi-quantitatively analyzed by densitometry. Ubiquitin signals larger than 50 kDa were considered as poly-Ubiquitination, while under 50 kDa as oligo-Ubiquitination [ 25 ]. ( B ) CDK18-FLAG kinase dead (K150R) or constitutively active (S12D)-encoding constructs were expressed in MCD4 cells, immunoprecipitated through their Flag tags and the interactomes analysed by mass-spectrometry. The x -axis indicates the abundance ratios of the identified interacting proteins of K150R (negative values) versus S12D (positive values). The y -axis represents the significance of a two-sample moderated t-test by which the proteins were quantified. The dashed line indicates the 5% FDR cut-off based on Benjamini–Hochberg corrected p -values. The identified STUB1 (green) and protein phosphatases (PP, red) subunits are highlighted. ( C ) MCD4 cells were transfected with NT or STUB1 siRNA, lysed and subjected to immunoprecipitation (IP) with anti-AQP2 or control (IgG) antibodies. AQP2, CDK18, STUB1, and in the input samples HSP90 as loading control were detected by Western blotting. Shown are representative results and the densitometric semi-quantitative analysis from n = 4 independent experiments. Ubiquitin (Ubi) signals larger than 50 kDa were considered poly-Ubiquitination, while the ones under 50 kDa were defined as oligo-Ubiquitination [ 25 ]. ( D ) Detection of AQP2 (green) by immunofluorescence microscopic analysis of MCD4 cells transfected with siNT or STUB1 siRNA, and stimulated with forskolin (Fsk) or vehicle. Nuclei (blue) were stained with DAPI; n = 3; scale bar, 50 µm). ( E ) MCD4 cells were left untransfected or transfected with control siNT or CDK18 siRNA, and stimulated with forskolin (Fsk) or vehicle. Representative Western blots of the indicated proteins including the detection of cadherin (pan-Cadherin) as a loading control is shown ( n = 4 independent experiments). The signals were semi-quantitatively analyzed by densitometry. ( F ) The STUB1 mRNA expression in the samples indicated in ( E ) was evaluated by sq-PCR ( n ≥ 4 per condition). ( G ) MCD4 cells were transfected with either siNT or CDK18 siRNA, and treated with the proteasome inhibitor MG132 or vehicle as a control. The cells were lysed and the indicated proteins were detected by Western blotting. Shown is a representative of n = 5 independent experiments. The signals were semi-quantitatively analyzed by densitometry. ( H ) Mouse primary inner medullary collecting duct cells were transfected with either siNT or CDK18 siRNA. STUB1, CDK18 and HSP90 as a loading control were detected by Western blotting. Shown is the result from n = 5 independent experiments. The signals were semi-quantitatively analyzed by densitometry. Statistically significant differences are indicated, * p

    Journal: Cells

    Article Title: Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

    doi: 10.3390/cells9030673

    Figure Lengend Snippet: CDK18 regulates STUB1 to control AQP2 ubiquitination, abundance and localisation. ( A ) MCD4 cells were transfected with control non-targeting (siNT) or CDK18 siRNA, lysed and AQP2 was immunoprecipitated (IP). As a control, unrelated IgG was used (IgG). AQP2 and co-immunoprecipitated ubiquitin (Ubi) were detected by Western blotting (WB). Shown are a representative result from n = 4 independent experiments including the input samples where HSP90 was used as a loading control. The signals were semi-quantitatively analyzed by densitometry. Ubiquitin signals larger than 50 kDa were considered as poly-Ubiquitination, while under 50 kDa as oligo-Ubiquitination [ 25 ]. ( B ) CDK18-FLAG kinase dead (K150R) or constitutively active (S12D)-encoding constructs were expressed in MCD4 cells, immunoprecipitated through their Flag tags and the interactomes analysed by mass-spectrometry. The x -axis indicates the abundance ratios of the identified interacting proteins of K150R (negative values) versus S12D (positive values). The y -axis represents the significance of a two-sample moderated t-test by which the proteins were quantified. The dashed line indicates the 5% FDR cut-off based on Benjamini–Hochberg corrected p -values. The identified STUB1 (green) and protein phosphatases (PP, red) subunits are highlighted. ( C ) MCD4 cells were transfected with NT or STUB1 siRNA, lysed and subjected to immunoprecipitation (IP) with anti-AQP2 or control (IgG) antibodies. AQP2, CDK18, STUB1, and in the input samples HSP90 as loading control were detected by Western blotting. Shown are representative results and the densitometric semi-quantitative analysis from n = 4 independent experiments. Ubiquitin (Ubi) signals larger than 50 kDa were considered poly-Ubiquitination, while the ones under 50 kDa were defined as oligo-Ubiquitination [ 25 ]. ( D ) Detection of AQP2 (green) by immunofluorescence microscopic analysis of MCD4 cells transfected with siNT or STUB1 siRNA, and stimulated with forskolin (Fsk) or vehicle. Nuclei (blue) were stained with DAPI; n = 3; scale bar, 50 µm). ( E ) MCD4 cells were left untransfected or transfected with control siNT or CDK18 siRNA, and stimulated with forskolin (Fsk) or vehicle. Representative Western blots of the indicated proteins including the detection of cadherin (pan-Cadherin) as a loading control is shown ( n = 4 independent experiments). The signals were semi-quantitatively analyzed by densitometry. ( F ) The STUB1 mRNA expression in the samples indicated in ( E ) was evaluated by sq-PCR ( n ≥ 4 per condition). ( G ) MCD4 cells were transfected with either siNT or CDK18 siRNA, and treated with the proteasome inhibitor MG132 or vehicle as a control. The cells were lysed and the indicated proteins were detected by Western blotting. Shown is a representative of n = 5 independent experiments. The signals were semi-quantitatively analyzed by densitometry. ( H ) Mouse primary inner medullary collecting duct cells were transfected with either siNT or CDK18 siRNA. STUB1, CDK18 and HSP90 as a loading control were detected by Western blotting. Shown is the result from n = 5 independent experiments. The signals were semi-quantitatively analyzed by densitometry. Statistically significant differences are indicated, * p

    Article Snippet: CDK18 siRNA (#M-040145-01-0005/18557)), STUB1 (#M-007201-02-0005) and non-targeting siRNA (siNT; NT#2) (against firefly luciferase; #D-001206-14-20) were purchased as siGENOME SMART pools (Thermo Fisher).

    Techniques: Transfection, Immunoprecipitation, Western Blot, Construct, Mass Spectrometry, Immunofluorescence, Staining, Expressing, Polymerase Chain Reaction

    ( A ) MCD4 cells were lysed and PKA activity was determined by measuring its ability to phosphorylate a substrate peptide (PepTag A1). Shown is a representative agarose gels from n = 4 independent experiments with PKA-phosphorylated (pPept) and non-phosphorylated (Pept) PepTag A1 peptide. The amounts of each peptide were semi-quantitatively analysed and relative PKA activity was expressed as the ratio of phosphorylated to non-phosphorylated peptides. ( B ) MCD4 cells were transfected with siNT or CDK18 siRNA, and stimulated with forskolin (Fsk). Phosphatase activity was assayed using para-nitrophenyl-phosphate (pNPP; n = 4). Statistically significant differences are indicated, * p

    Journal: Cells

    Article Title: Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

    doi: 10.3390/cells9030673

    Figure Lengend Snippet: ( A ) MCD4 cells were lysed and PKA activity was determined by measuring its ability to phosphorylate a substrate peptide (PepTag A1). Shown is a representative agarose gels from n = 4 independent experiments with PKA-phosphorylated (pPept) and non-phosphorylated (Pept) PepTag A1 peptide. The amounts of each peptide were semi-quantitatively analysed and relative PKA activity was expressed as the ratio of phosphorylated to non-phosphorylated peptides. ( B ) MCD4 cells were transfected with siNT or CDK18 siRNA, and stimulated with forskolin (Fsk). Phosphatase activity was assayed using para-nitrophenyl-phosphate (pNPP; n = 4). Statistically significant differences are indicated, * p

    Article Snippet: CDK18 siRNA (#M-040145-01-0005/18557)), STUB1 (#M-007201-02-0005) and non-targeting siRNA (siNT; NT#2) (against firefly luciferase; #D-001206-14-20) were purchased as siGENOME SMART pools (Thermo Fisher).

    Techniques: Activity Assay, Transfection

    STUB1 organizes a signalosome comprising CDK18, AQP2 and PKA and functions as an A-kinase anchoring protein (AKAP). ( A ) MCD4 cells were lysed and the lysates incubated with the peptide Ht31, which inhibits AKAP-PKA interactions, the inactive peptide Ht31-PP or vehicle and subjected to cAMP-agarose pull down (PD). As negative control, the pull down samples (PD) were incubated with an excess of cAMP. The indicated proteins in the pull down and input samples were detected by Western blotting and the signals were densitometrically evaluated. Shown are representative results from n = 4 independent experiments. ( B ) MCD4 cells were transfected with siRNA against STUB1 or control non-targeting (siNT) and subjected to cAMP-agarose pull down. As a control, the precipitates (PD) were incubated with an excess of cAMP. The indicated proteins were detected by Western blotting and the signals densitometrically evaluated shown are representative results from n = 5 independent experiments. ( C ) MCD4 were lysed and the lysates were left untreated, incubated with the peptide Ht31 or PP-Ht31. STUB1 was immunoprecipitated (IP). As control, precipitations were carried out with unrelated IgG. The indicated proteins in the precipitates and input samples were analyzed by Western blotting. HSP90 served as a loading control. Shown are representative results from n = 4 independent experiments. The signals were semi-quantitatively analysed by densitometry. ( D ) Murine STUB1 was spot-synthesized as 20-mer peptides with 5 residues offset. Spots 1–5 cover amino acids (Aa) 150–190. The spots were incubated with P 32 -labeled recombinant human RIIα subunits of PKA in the absence or presence of the peptide L314E, which inhibits AKAP-PKA interactions. A representative overlay from n = 4 independent experiments is shown. The semi-quantitative densitometric analysis is shown. ( E ) MCD4 cells were left untreated or stimulated with forskolin (Fsk) and STUB1 was immunoprecipitated. As a control, unrelated IgG (IgG) was used. The indicated proteins in the precipitates and the input samples were detected by Western blotting. Shown are representative results from n = 3 independent experiments. The signals were semi-quantitatively analyzed by densitometry. cg: complex glycosylated, hm: high-mannose, ng: non-glycosylated AQP2. Statistically significant differences are indicated, * p

    Journal: Cells

    Article Title: Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

    doi: 10.3390/cells9030673

    Figure Lengend Snippet: STUB1 organizes a signalosome comprising CDK18, AQP2 and PKA and functions as an A-kinase anchoring protein (AKAP). ( A ) MCD4 cells were lysed and the lysates incubated with the peptide Ht31, which inhibits AKAP-PKA interactions, the inactive peptide Ht31-PP or vehicle and subjected to cAMP-agarose pull down (PD). As negative control, the pull down samples (PD) were incubated with an excess of cAMP. The indicated proteins in the pull down and input samples were detected by Western blotting and the signals were densitometrically evaluated. Shown are representative results from n = 4 independent experiments. ( B ) MCD4 cells were transfected with siRNA against STUB1 or control non-targeting (siNT) and subjected to cAMP-agarose pull down. As a control, the precipitates (PD) were incubated with an excess of cAMP. The indicated proteins were detected by Western blotting and the signals densitometrically evaluated shown are representative results from n = 5 independent experiments. ( C ) MCD4 were lysed and the lysates were left untreated, incubated with the peptide Ht31 or PP-Ht31. STUB1 was immunoprecipitated (IP). As control, precipitations were carried out with unrelated IgG. The indicated proteins in the precipitates and input samples were analyzed by Western blotting. HSP90 served as a loading control. Shown are representative results from n = 4 independent experiments. The signals were semi-quantitatively analysed by densitometry. ( D ) Murine STUB1 was spot-synthesized as 20-mer peptides with 5 residues offset. Spots 1–5 cover amino acids (Aa) 150–190. The spots were incubated with P 32 -labeled recombinant human RIIα subunits of PKA in the absence or presence of the peptide L314E, which inhibits AKAP-PKA interactions. A representative overlay from n = 4 independent experiments is shown. The semi-quantitative densitometric analysis is shown. ( E ) MCD4 cells were left untreated or stimulated with forskolin (Fsk) and STUB1 was immunoprecipitated. As a control, unrelated IgG (IgG) was used. The indicated proteins in the precipitates and the input samples were detected by Western blotting. Shown are representative results from n = 3 independent experiments. The signals were semi-quantitatively analyzed by densitometry. cg: complex glycosylated, hm: high-mannose, ng: non-glycosylated AQP2. Statistically significant differences are indicated, * p

    Article Snippet: CDK18 siRNA (#M-040145-01-0005/18557)), STUB1 (#M-007201-02-0005) and non-targeting siRNA (siNT; NT#2) (against firefly luciferase; #D-001206-14-20) were purchased as siGENOME SMART pools (Thermo Fisher).

    Techniques: Incubation, Negative Control, Western Blot, Transfection, Immunoprecipitation, Synthesized, Labeling, Recombinant

    A cytosolic, STUB1-independent pool of PKA phosphorylates CDK18. ( A ) MCD4 cells were transfected with non-targeting (siNT) or STUB1 siRNA and left untreated or were treated with forskolin (Fsk) in the absence or ( B ) presence of a stearate-coupled, membrane-permeant version of the peptide Ht31, which inhibits AKAP-PKA interactions. CDK18 was immunoprecipitated (IP). As control, precipitations were carried out with unrelated IgG. The indicated proteins were detected by Western blotting. Phosphorylated CDK18 (pSub PKA) was detected with an phospho-PKA substrate antibody and CDK18 with specific antibodies; in the input samples, CDK18, STUB1, as a control for the effects of AKAP-PKA disruption, GSK3β phosphorylated by PKA (pS9), and as a loading control HSP90. Shown are representative results from n = 4 independent experiments and the semi-quantitative densitometric analysis. ( C ) MCD4 cells were left untreated or transfected with control non-targeting (siNT) or STUB1 siRNA, and stimulated with forskolin (Fsk) or vehicle. The cells were lysed and the indicated proteins detected by Western blot (WB) analysis. The signals were densitometrically evaluated. Shown are representative results from n = 5 independent experiments. Statistically significant differences are indicated, * p

    Journal: Cells

    Article Title: Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

    doi: 10.3390/cells9030673

    Figure Lengend Snippet: A cytosolic, STUB1-independent pool of PKA phosphorylates CDK18. ( A ) MCD4 cells were transfected with non-targeting (siNT) or STUB1 siRNA and left untreated or were treated with forskolin (Fsk) in the absence or ( B ) presence of a stearate-coupled, membrane-permeant version of the peptide Ht31, which inhibits AKAP-PKA interactions. CDK18 was immunoprecipitated (IP). As control, precipitations were carried out with unrelated IgG. The indicated proteins were detected by Western blotting. Phosphorylated CDK18 (pSub PKA) was detected with an phospho-PKA substrate antibody and CDK18 with specific antibodies; in the input samples, CDK18, STUB1, as a control for the effects of AKAP-PKA disruption, GSK3β phosphorylated by PKA (pS9), and as a loading control HSP90. Shown are representative results from n = 4 independent experiments and the semi-quantitative densitometric analysis. ( C ) MCD4 cells were left untreated or transfected with control non-targeting (siNT) or STUB1 siRNA, and stimulated with forskolin (Fsk) or vehicle. The cells were lysed and the indicated proteins detected by Western blot (WB) analysis. The signals were densitometrically evaluated. Shown are representative results from n = 5 independent experiments. Statistically significant differences are indicated, * p

    Article Snippet: CDK18 siRNA (#M-040145-01-0005/18557)), STUB1 (#M-007201-02-0005) and non-targeting siRNA (siNT; NT#2) (against firefly luciferase; #D-001206-14-20) were purchased as siGENOME SMART pools (Thermo Fisher).

    Techniques: Transfection, Immunoprecipitation, Western Blot

    CDK18 is necessary for the cAMP-induced redistribution of AQP2 from intracellular vesicles to the plasma membrane. ( A ) Schematic representation of the Kinome-wide siRNA screening approach. MCD4 cells were seeded in 384-well microtiter plates and the expression of 719 kinases was knocked down each with a pool of four siRNAs. The effects of the knockdown on the localization of AQP2 were detected with specific anti-AQP2 and secondary Cy3-coupled antibodies and automated immunofluorescence microscopic analysis. Image analysis was carried out with CellProfiler and KNIME software. ( B ) MCD4 cells were treated with 50 nM non-targeting siRNA (siNT), a pool of four different or a single CDK18 siRNA. The cells were treated with forskolin (Fsk; 30 µM, 60 min) or were left unstimulated (control) and the localization of AQP2 was analyzed with a confocal laser scanning microscope (40× magnification). AQP2 is in green and nuclei are in blue. Shown are representative images from n ≥ 3 independent experiments per condition; scale bar, 50 µm. ( C ) The efficacy of the CDK18 knockdown was evaluated by Western blot analysis. CDK18 and as a loading control Pan-cadherin were detected. Signals were quantified by densitometric analysis. Statistical analysis was performed using the unpaired t-test, significant differences are indicated, * p ≤ 0.05, ** p ≤ 0.01. Mean ± SEM are plotted, n = 3–6 independent experiments per condition.

    Journal: Cells

    Article Title: Cyclin-Dependent Kinase 18 Controls Trafficking of Aquaporin-2 and Its Abundance through Ubiquitin Ligase STUB1, Which Functions as an AKAP

    doi: 10.3390/cells9030673

    Figure Lengend Snippet: CDK18 is necessary for the cAMP-induced redistribution of AQP2 from intracellular vesicles to the plasma membrane. ( A ) Schematic representation of the Kinome-wide siRNA screening approach. MCD4 cells were seeded in 384-well microtiter plates and the expression of 719 kinases was knocked down each with a pool of four siRNAs. The effects of the knockdown on the localization of AQP2 were detected with specific anti-AQP2 and secondary Cy3-coupled antibodies and automated immunofluorescence microscopic analysis. Image analysis was carried out with CellProfiler and KNIME software. ( B ) MCD4 cells were treated with 50 nM non-targeting siRNA (siNT), a pool of four different or a single CDK18 siRNA. The cells were treated with forskolin (Fsk; 30 µM, 60 min) or were left unstimulated (control) and the localization of AQP2 was analyzed with a confocal laser scanning microscope (40× magnification). AQP2 is in green and nuclei are in blue. Shown are representative images from n ≥ 3 independent experiments per condition; scale bar, 50 µm. ( C ) The efficacy of the CDK18 knockdown was evaluated by Western blot analysis. CDK18 and as a loading control Pan-cadherin were detected. Signals were quantified by densitometric analysis. Statistical analysis was performed using the unpaired t-test, significant differences are indicated, * p ≤ 0.05, ** p ≤ 0.01. Mean ± SEM are plotted, n = 3–6 independent experiments per condition.

    Article Snippet: CDK18 siRNA (#M-040145-01-0005/18557)), STUB1 (#M-007201-02-0005) and non-targeting siRNA (siNT; NT#2) (against firefly luciferase; #D-001206-14-20) were purchased as siGENOME SMART pools (Thermo Fisher).

    Techniques: Expressing, Immunofluorescence, Software, Laser-Scanning Microscopy, Western Blot