reaction buffer  (Millipore)


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  • 99
    Name:
    Dimethylformamide
    Description:
    Pharmaceutical secondary standards for application in quality control provide pharma laboratories and manufacturers with a convenient and cost effective alternative to the preparation of in house working standards
    Catalog Number:
    phr1553
    Price:
    None
    Applications:
    Solvent for many hydrophobic organic compounds.
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    Millipore reaction buffer
    Dimethylformamide
    Pharmaceutical secondary standards for application in quality control provide pharma laboratories and manufacturers with a convenient and cost effective alternative to the preparation of in house working standards
    https://www.bioz.com/result/reaction buffer/product/Millipore
    Average 99 stars, based on 398 article reviews
    Price from $9.99 to $1999.99
    reaction buffer - by Bioz Stars, 2020-09
    99/100 stars

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    High Performance Liquid Chromatography:

    Article Title: Preparation of polylactide-co-glycolide nanoparticles incorporating celecoxib and their antitumor activity against brain tumor cells
    Article Snippet: .. Dimethylformamide, dimethylacetamide, tetrahydrofuran, dimethylsulfoxide, 1,4-dioxane, and acetone of high-pressure liquid chromatography grade were purchased from Sigma-Aldrich Chemical Company Ltd (St Louis, MO). ..

    MTT Assay:

    Article Title: Polyamidoamine (PAMAM) Dendrimers Modified with Cathepsin-B Cleavable Oligopeptides for Enhanced Gene Delivery
    Article Snippet: .. Deuterium oxide (D2 O), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), piperidine, triisopropylsilane (TIS), trifluoroacetic acid (TFA), cathepsin B (from human liver), thiazolyl blue tetrazolium bromide (MTT), ethidium bromide (EtBr), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), PAMAM dendrimer (ethylene diamine core, generation 4.0 solution), and N ,N -diisopropylethylamine (DIPEA) were purchased from Sigma Aldrich (St. Louis, MO, USA). .. Fmoc-Phe-OH, N -α-Fmoc-N -g -(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl(pbf))-l -arginine, Fmoc-Leu-OH, and 1-hydroxybenzotriazole hydrate (HOBt) were obtained from Anaspec (Fremont, CA, USA).

    Exclusion Assay:

    Article Title: Dual Effects of N,N-dimethylformamide on Cell Proliferation and Apoptosis in Breast Cancer
    Article Snippet: .. N,N-dimethylformamide Treatment and Trypan Blue Dye Exclusion Assay Both MCF-7 and MCF-12A cells were treated with DMF (D4551-250ML; Sigma) at the dose of 0, 0.1, 1, 10, 31.25, 62.5, 100, 125, 250, and 500 mM for 24, 48, and 72 hours, respectively. .. Cells were then incubated with trypan blue dye (Cat# KGY015; Keygen, Nanjing, Jiangsu, China) for 15 minutes and subsequently washed 3 times with phosphate-buffered saline (PBS).

    other:

    Article Title: Using Triple Helix Forming Peptide Nucleic Acids for Sequence-selective Recognition of Double-stranded RNA
    Article Snippet: Deblocking solution (20% (v/v) piperidine in dimethylformamide): mix 20 mL of piperidine (biotech grade, Aldrich) and 80 mL of anhydrous dimethylformamide.

    Article Title: The Phosphine Oxide Route toward Lead Halide Perovskite Nanocrystals
    Article Snippet: Acetone (99.5%), cesium carbonate (Cs2 CO3 , 99%), N ,N -dimethylformamide (99.8%, anhydrous), dimethyl sulfoxide (99.9%), oleylamine (70%, OlAm), oleic acid (90%, OA), diisooctylphosphinic acid (90%, DOPA), 1-octadecene (90%), toluene (≥99.7%), toluene-d 8 (d -toluene, 99.8 atom % D), and PbBr2 (≥98%) were purchased from Sigma-Aldrich.

    Purification:

    Article Title: Multivalency Enables Dynamic Supramolecular Host–Guest Hydrogel Formation
    Article Snippet: .. Materials All materials were acquired from suppliers indicated and used without further purification unless stated otherwise: toluene ( > 99.8%, Acros Organics), 2-arm PEG–OH (4.6 kDa, Sigma-Aldrich, PEG2-OH), 4-arm PEG–OH (10 kDa, 97.5%, Creative PEGWorks, PEG4-OH), 8-arm PEG–OH (hexaglycerol core, 20 kDa, 99.1%, Creative PEGWorks, PEG8-OH), 1-adamantane methylamine ( > 98.0%, TCI Europe, ADA), triethylamine ( > 99%, Merck, Et3 N), 1,4-dioxane (99.8%, Sigma-Aldrich), 1,6-hexadiamine (98%, Sigma-Aldrich), 4-toluenesulfyl chloride ( > 98%, Sigma-Aldrich, OTs) anhydrous chloroform ( > 99%, Sigma-Aldrich), anhydrous dimethylformamide (99.8%, Sigma-Aldrich, DMF), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (98+%, VWR, EDC-HCl), N -hydroxysulfosuccinimide sodium salt (≥98%, Sigma-Aldrich, sulfo-NHS), β-cyclodextrin (99%, Sigma-Aldrich, CD), carbonyl diimidazole ( > 90%, Sigma-Aldrich, CDI), deuterated chloroform ( > 99.8%, Sigma-Aldrich, CDCl3 ), deuterium oxide (99.9%, Sigma-Aldrich, D2 O), Snakeskin MWCO 10,000 dialysis tubes (Thermo Scientific U.S.A.), diethyl ether ( > 99%, VWR). .. Sodium hydride (NaH, 60 wt % in mineral oil) was washed with hexane and tetrahydrofuran (THF) under an argon atmosphere.

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  • 85
    Millipore erk1 reaction buffer
    Expression of a constitutive active form of MEK1 induces NIC1 post-translational modifications when expressed in HEK293T cells. A . HEK293T were transfected with pLIA-NIC1 (MYC-tagged) construct together with a cDNA encoding a wild-type (WT) or constitutive active (CA) version of MEK1 (HA-tagged). Cells were lysed 24h post-transfection. NIC1 expression was analysed by western blot using an anti-MYC antibody. The expression level of the exogenous MEK1 was evaluated by western blot using an anti-HA antibody. Dual-phosphorylated and total <t>ERK1/2</t> expression levels were assessed using specific antibodies. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. B . HEK293T were transfected with pLIA-NIC1 construct together with a cDNA encoding MEK1 WT or MEK1 CA. Cells were lysed and NIC1 was immunoprecipitated (IP NIC1) using an anti-MYC antibody. Phosphatase assays were then performed (+) using the lambda phosphatase (λ phosphatase). The phosphorylated forms of NIC1 are denoted pNIC1. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. NIC1 expression was analysed by western blot using an anti-MYC antibody. C . NIC1 was immunoprecipitated (IP NIC1) from pLIA-NIC1 transfected HEK293T using an anti-MYC antibody. Kinase assays were then performed as described in experimental procedures. The autoradiography depicting phosphorylated NIC1 (pNIC1) is shown. Following electrotransfer of the gel, the amount of NIC1 immunoprecipitated was confirmed by western blot (WB) using an anti-MYC antibody.
    Erk1 Reaction Buffer, supplied by Millipore, 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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    86
    Millipore msk1 reaction buffer
    HP1γ bridges <t>MSK1</t> at the chromatin and regulates the expression of specific genes WT and HP1γ null mouse fibroblasts were stimulated by PMA at the indicated times. Nuclear extract were processed as indicated in Materials and Methods. Western blots were performed with anti-MSK1, anti-HP1γS83p, and anti-histone H3 antibodies. HeLa cells were transfected with siHP1γ or, as a control, siGAPDH and stimulated by PMA (40 min). ChIPs were performed using anti-HP1γ or anti-MSK1 antibodies, or unrelated mouse IgGs as a control. Results are expressed as fold induction versus IgG for each point (** P
    Msk1 Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msk1 reaction buffer/product/Millipore
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    91
    Millipore calpain reaction buffer
    Schematic diagram showing the relationship of <t>calpain/NF-κB/inflammation/NVU</t> damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.
    Calpain Reaction Buffer, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore complete embryonic stem es cell media
    The gene expression profile of the pancreatic <t>stem</t> cells cultured under condition #3. To investigate the gene expression profile of HN#101 cells cultured for 2 months under culture condition #3, an RT-PCR analysis of endodermal/pancreatic progenitor <t>cell</t> markers was performed. Feeder, feeder cells (STO cells, negative control); PDL 50, HN#101 cells at PDL 50 under culture condition #1; ESM + Feeder, HN#101 cells in <t>ES</t> <t>media</t> (ESM) at PDL 100 under culture condition #3; DE, differentiated cells at the definitive endoderm stage derived from ES cells (positive control); GTE, differentiated cells at the gut tube endoderm stage derived from ES cells (positive control); PP, differentiated cells at the pancreatic progenitor stage derived from ES cells (positive control); Sox17, sex-determining region Y box 17; Foxa2, forkhead box A2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Hnf, hepatocyte growth factor; Pdx1, pancreatic and duodenal homeobox factor 1.
    Complete Embryonic Stem Es Cell Media, supplied by Millipore, 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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    Expression of a constitutive active form of MEK1 induces NIC1 post-translational modifications when expressed in HEK293T cells. A . HEK293T were transfected with pLIA-NIC1 (MYC-tagged) construct together with a cDNA encoding a wild-type (WT) or constitutive active (CA) version of MEK1 (HA-tagged). Cells were lysed 24h post-transfection. NIC1 expression was analysed by western blot using an anti-MYC antibody. The expression level of the exogenous MEK1 was evaluated by western blot using an anti-HA antibody. Dual-phosphorylated and total ERK1/2 expression levels were assessed using specific antibodies. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. B . HEK293T were transfected with pLIA-NIC1 construct together with a cDNA encoding MEK1 WT or MEK1 CA. Cells were lysed and NIC1 was immunoprecipitated (IP NIC1) using an anti-MYC antibody. Phosphatase assays were then performed (+) using the lambda phosphatase (λ phosphatase). The phosphorylated forms of NIC1 are denoted pNIC1. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. NIC1 expression was analysed by western blot using an anti-MYC antibody. C . NIC1 was immunoprecipitated (IP NIC1) from pLIA-NIC1 transfected HEK293T using an anti-MYC antibody. Kinase assays were then performed as described in experimental procedures. The autoradiography depicting phosphorylated NIC1 (pNIC1) is shown. Following electrotransfer of the gel, the amount of NIC1 immunoprecipitated was confirmed by western blot (WB) using an anti-MYC antibody.

    Journal: PLoS ONE

    Article Title: The MEK/ERK Pathway Promotes NOTCH Signalling in Pancreatic Cancer Cells

    doi: 10.1371/journal.pone.0085502

    Figure Lengend Snippet: Expression of a constitutive active form of MEK1 induces NIC1 post-translational modifications when expressed in HEK293T cells. A . HEK293T were transfected with pLIA-NIC1 (MYC-tagged) construct together with a cDNA encoding a wild-type (WT) or constitutive active (CA) version of MEK1 (HA-tagged). Cells were lysed 24h post-transfection. NIC1 expression was analysed by western blot using an anti-MYC antibody. The expression level of the exogenous MEK1 was evaluated by western blot using an anti-HA antibody. Dual-phosphorylated and total ERK1/2 expression levels were assessed using specific antibodies. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. B . HEK293T were transfected with pLIA-NIC1 construct together with a cDNA encoding MEK1 WT or MEK1 CA. Cells were lysed and NIC1 was immunoprecipitated (IP NIC1) using an anti-MYC antibody. Phosphatase assays were then performed (+) using the lambda phosphatase (λ phosphatase). The phosphorylated forms of NIC1 are denoted pNIC1. The MEK1 CA-induced higher molecular weight form of NIC1 is indicated by an asterisk *. NIC1 expression was analysed by western blot using an anti-MYC antibody. C . NIC1 was immunoprecipitated (IP NIC1) from pLIA-NIC1 transfected HEK293T using an anti-MYC antibody. Kinase assays were then performed as described in experimental procedures. The autoradiography depicting phosphorylated NIC1 (pNIC1) is shown. Following electrotransfer of the gel, the amount of NIC1 immunoprecipitated was confirmed by western blot (WB) using an anti-MYC antibody.

    Article Snippet: The beads were then equally split in two Eppendorf tubes and incubated 30min at 30°C in ERK1 reaction buffer supplemented with 10mM magnesium acetate, 0.1mM ATP and 2μCi [γ-32 P]ATP containing or not 0.1μg of active ERK1 (Millipore).

    Techniques: Expressing, Transfection, Construct, Western Blot, Molecular Weight, Immunoprecipitation, Autoradiography, Electrotransfer

    HP1γ bridges MSK1 at the chromatin and regulates the expression of specific genes WT and HP1γ null mouse fibroblasts were stimulated by PMA at the indicated times. Nuclear extract were processed as indicated in Materials and Methods. Western blots were performed with anti-MSK1, anti-HP1γS83p, and anti-histone H3 antibodies. HeLa cells were transfected with siHP1γ or, as a control, siGAPDH and stimulated by PMA (40 min). ChIPs were performed using anti-HP1γ or anti-MSK1 antibodies, or unrelated mouse IgGs as a control. Results are expressed as fold induction versus IgG for each point (** P

    Journal: The EMBO Journal

    Article Title: Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF

    doi: 10.15252/embj.201489244

    Figure Lengend Snippet: HP1γ bridges MSK1 at the chromatin and regulates the expression of specific genes WT and HP1γ null mouse fibroblasts were stimulated by PMA at the indicated times. Nuclear extract were processed as indicated in Materials and Methods. Western blots were performed with anti-MSK1, anti-HP1γS83p, and anti-histone H3 antibodies. HeLa cells were transfected with siHP1γ or, as a control, siGAPDH and stimulated by PMA (40 min). ChIPs were performed using anti-HP1γ or anti-MSK1 antibodies, or unrelated mouse IgGs as a control. Results are expressed as fold induction versus IgG for each point (** P

    Article Snippet: Control reactions were performed in MSK1 reaction buffer on 150 ng of phosphorylated GST-ERK1 (14-439; Millipore).

    Techniques: Expressing, Western Blot, Transfection

    Model: OspF targets the nuclear MAPK/HP1γ signaling pathway and reveals the function of the MSK1/HP1γ cross talk In unstimulated cells (ns), HP1 is brought to the promoter by histone H3K9 methylation and acts as a transcriptional repressor. Shigella flexneri infection results in activation of the MAPK/MSK1 pathway, but this activation is rapidly dampened by OspF that dephosphorylates ERK. The combined effect of these two events is a moderate transcriptional activation of IL8 . This causes HP1γ to leave the promoter, while inactivation of ERK prevents it from getting phosphorylated and join the transcription machinery. Consequently, accumulation of HP1γ at the IL8 gene drops. Upon ospF Shigella infection or treatment with PMA, the MAPK nuclear pathway is fully triggered, levels of histone H3 marks permissive for transcription are increased (H3S10), and the transcription machinery is recruited. Activation of the MAPK nuclear pathway induces HP1γ phosphorylation and allows for its recruitment to IL8 and other similarly regulated immune genes. In MEF KO cells, the loss of HP1γ eliminates an important checkpoint in the control of immune gene activation, leading to a deregulated possibly excessive gene expression in response to MAPK activation.

    Journal: The EMBO Journal

    Article Title: Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF

    doi: 10.15252/embj.201489244

    Figure Lengend Snippet: Model: OspF targets the nuclear MAPK/HP1γ signaling pathway and reveals the function of the MSK1/HP1γ cross talk In unstimulated cells (ns), HP1 is brought to the promoter by histone H3K9 methylation and acts as a transcriptional repressor. Shigella flexneri infection results in activation of the MAPK/MSK1 pathway, but this activation is rapidly dampened by OspF that dephosphorylates ERK. The combined effect of these two events is a moderate transcriptional activation of IL8 . This causes HP1γ to leave the promoter, while inactivation of ERK prevents it from getting phosphorylated and join the transcription machinery. Consequently, accumulation of HP1γ at the IL8 gene drops. Upon ospF Shigella infection or treatment with PMA, the MAPK nuclear pathway is fully triggered, levels of histone H3 marks permissive for transcription are increased (H3S10), and the transcription machinery is recruited. Activation of the MAPK nuclear pathway induces HP1γ phosphorylation and allows for its recruitment to IL8 and other similarly regulated immune genes. In MEF KO cells, the loss of HP1γ eliminates an important checkpoint in the control of immune gene activation, leading to a deregulated possibly excessive gene expression in response to MAPK activation.

    Article Snippet: Control reactions were performed in MSK1 reaction buffer on 150 ng of phosphorylated GST-ERK1 (14-439; Millipore).

    Techniques: Methylation, Infection, Activation Assay, Expressing

    MSK1 forms a molecular complex with the phosphorylated pool of HP1γ in cells PMA stimulation leads to the formation of molecular tri-complex between the HP1γ and the ERK/MSK1 kinases. HeLa cells were stimulation by PMA (60 min), and cellular extracts were immunoprecipitated with mouse IgG as a negative control or anti-MSK1S360p or anti-HP1γ antibodies. Western blots were performed with anti-MSK1, anti-ERK, and anti-HP1γ antibodies. The phosphorylated pool of HP1γ pulls down the ERK kinase. Cellular extracts from HeLa cells were immunoprecipitated with mouse IgG as a negative control or anti-HP1γS83p, or anti-HP1γ antibodies. Western blots were performed with anti-ERK and anti-HP1γS83p antibodies. Confocal microscopy showing high coincidence of immunostaining between HP1γS83p and active MSK1. Immunofluorescence was performed with anti-HP1γS83p (red) and anti-MSK1S360p (green) antibodies. Confocal microscopy showing the immunostaining between HP1γS83p (red) and MSK1 upon infection with the WT and ospF mutant strains (green). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF

    doi: 10.15252/embj.201489244

    Figure Lengend Snippet: MSK1 forms a molecular complex with the phosphorylated pool of HP1γ in cells PMA stimulation leads to the formation of molecular tri-complex between the HP1γ and the ERK/MSK1 kinases. HeLa cells were stimulation by PMA (60 min), and cellular extracts were immunoprecipitated with mouse IgG as a negative control or anti-MSK1S360p or anti-HP1γ antibodies. Western blots were performed with anti-MSK1, anti-ERK, and anti-HP1γ antibodies. The phosphorylated pool of HP1γ pulls down the ERK kinase. Cellular extracts from HeLa cells were immunoprecipitated with mouse IgG as a negative control or anti-HP1γS83p, or anti-HP1γ antibodies. Western blots were performed with anti-ERK and anti-HP1γS83p antibodies. Confocal microscopy showing high coincidence of immunostaining between HP1γS83p and active MSK1. Immunofluorescence was performed with anti-HP1γS83p (red) and anti-MSK1S360p (green) antibodies. Confocal microscopy showing the immunostaining between HP1γS83p (red) and MSK1 upon infection with the WT and ospF mutant strains (green). Source data are available online for this figure.

    Article Snippet: Control reactions were performed in MSK1 reaction buffer on 150 ng of phosphorylated GST-ERK1 (14-439; Millipore).

    Techniques: Immunoprecipitation, Negative Control, Western Blot, Confocal Microscopy, Immunostaining, Immunofluorescence, Infection, Mutagenesis

    The MSK1 kinase drives HP1γS83p formation in Shigella -infected cells A OspF leads to the accumulation of a dephosphorylated and inactive form of MSK1 into Shigella -infected cells. HeLa cells were infected with the Shigella flexneri invasive (WT) or the ospF -deficient ( ospF ) strains at the indicated times or PMA (60 min). Western blots were performed with anti-MSK1S360p or MSK1 antibodies. B The MSK1 kinase modulates the HP1γS83p signal in Shigella -infected cells. Primary MEFs WT and MSK1/2 double null (MEF MSK1/2 −/− ) were infected with the Shigella flexneri invasive strain (WT) or the OspF-deficient strain ( ospF ) at the indicated times. Western blots were performed with anti-HP1γS83p, anti-HP1γ, anti-phospho-ERK, and anti-actin antibodies. Lower panel: Densitometric quantification of the signal obtained by Western blot. For each time point, the results are expressed as the level of HP1γS83p signal related to the total amount of HP1γ. C, D Kinase assays showing that MSK1 directly phosphorylates HP1γ at the S83 residue. Purified active MSK1 was incubated with histones, GST or GST-HP1α, GST-HP1β, GST-HP1γ or the indicated GST-HP1γ mutant fusion proteins. Kinase assay was performed at 30°C during 1 h in the presence of [γ- 32 P]-ATP followed by autoradiography. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF

    doi: 10.15252/embj.201489244

    Figure Lengend Snippet: The MSK1 kinase drives HP1γS83p formation in Shigella -infected cells A OspF leads to the accumulation of a dephosphorylated and inactive form of MSK1 into Shigella -infected cells. HeLa cells were infected with the Shigella flexneri invasive (WT) or the ospF -deficient ( ospF ) strains at the indicated times or PMA (60 min). Western blots were performed with anti-MSK1S360p or MSK1 antibodies. B The MSK1 kinase modulates the HP1γS83p signal in Shigella -infected cells. Primary MEFs WT and MSK1/2 double null (MEF MSK1/2 −/− ) were infected with the Shigella flexneri invasive strain (WT) or the OspF-deficient strain ( ospF ) at the indicated times. Western blots were performed with anti-HP1γS83p, anti-HP1γ, anti-phospho-ERK, and anti-actin antibodies. Lower panel: Densitometric quantification of the signal obtained by Western blot. For each time point, the results are expressed as the level of HP1γS83p signal related to the total amount of HP1γ. C, D Kinase assays showing that MSK1 directly phosphorylates HP1γ at the S83 residue. Purified active MSK1 was incubated with histones, GST or GST-HP1α, GST-HP1β, GST-HP1γ or the indicated GST-HP1γ mutant fusion proteins. Kinase assay was performed at 30°C during 1 h in the presence of [γ- 32 P]-ATP followed by autoradiography. Source data are available online for this figure.

    Article Snippet: Control reactions were performed in MSK1 reaction buffer on 150 ng of phosphorylated GST-ERK1 (14-439; Millipore).

    Techniques: Infection, Western Blot, Purification, Incubation, Mutagenesis, Kinase Assay, Autoradiography

    The virulence effector OspF blocks formation of HP1γS83p in Shigella- infected cells Shigella inhibits PMA-induced formation of HP1γS83p. HeLa cells were infected with the Shigella flexneri invasive strain (WT) and stimulated or not by PMA at the indicated times. Western blots were performed with anti-HP1γS83p, anti-HP1γ, and anti-actin antibodies. Pharmacological inhibition of the MAPK/MSK1 pathway blocks formation of HP1γS83p. HeLa cells were pretreated for 1 h with the MEK1 inhibitor U0126 or MSK1 inhibitor H89 and stimulated by PMA. Western blots were performed with anti-HP1γS83p, anti-HP1γ, anti-H3K9me2S10p, or anti-H3 antibodies. HeLa cells were infected with the Shigella flexneri invasive (WT) or the ospF -deficient ( ospF ) strains at the indicated time or stimulated by PMA (60 min). Western blots were performed with anti-HP1γS83p, anti-HP1γ, and anti-actin antibodies. Immunoprecipitation of endogenous HP1γ from HeLa cells overexpressing OspF. HeLa cells were transiently transfected with the indicated plasmids. Cellular extract were immunoprecipitated with mouse IgG as a negative control or HP1γ antibodies. Western blots were performed with OspF and HP1γ antibodies. The arrow indicates the OspF signal. Dephosphorylation at S83 in HP1γ immunoprecipitates containing OspF. HeLa cells were transiently transfected with the indicated plasmids and cellular extract immunoprecipitated with HP1γ antibody in the absence or the presence of DNase I. Western blots were performed with anti-HP1γS83p, anti-HP1γ, anti-OspF, or anti-ERK antibodies. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Shigella flexneri targets the HP1γ subcode through the phosphothreonine lyase OspF

    doi: 10.15252/embj.201489244

    Figure Lengend Snippet: The virulence effector OspF blocks formation of HP1γS83p in Shigella- infected cells Shigella inhibits PMA-induced formation of HP1γS83p. HeLa cells were infected with the Shigella flexneri invasive strain (WT) and stimulated or not by PMA at the indicated times. Western blots were performed with anti-HP1γS83p, anti-HP1γ, and anti-actin antibodies. Pharmacological inhibition of the MAPK/MSK1 pathway blocks formation of HP1γS83p. HeLa cells were pretreated for 1 h with the MEK1 inhibitor U0126 or MSK1 inhibitor H89 and stimulated by PMA. Western blots were performed with anti-HP1γS83p, anti-HP1γ, anti-H3K9me2S10p, or anti-H3 antibodies. HeLa cells were infected with the Shigella flexneri invasive (WT) or the ospF -deficient ( ospF ) strains at the indicated time or stimulated by PMA (60 min). Western blots were performed with anti-HP1γS83p, anti-HP1γ, and anti-actin antibodies. Immunoprecipitation of endogenous HP1γ from HeLa cells overexpressing OspF. HeLa cells were transiently transfected with the indicated plasmids. Cellular extract were immunoprecipitated with mouse IgG as a negative control or HP1γ antibodies. Western blots were performed with OspF and HP1γ antibodies. The arrow indicates the OspF signal. Dephosphorylation at S83 in HP1γ immunoprecipitates containing OspF. HeLa cells were transiently transfected with the indicated plasmids and cellular extract immunoprecipitated with HP1γ antibody in the absence or the presence of DNase I. Western blots were performed with anti-HP1γS83p, anti-HP1γ, anti-OspF, or anti-ERK antibodies. Source data are available online for this figure.

    Article Snippet: Control reactions were performed in MSK1 reaction buffer on 150 ng of phosphorylated GST-ERK1 (14-439; Millipore).

    Techniques: Infection, Western Blot, Inhibition, Immunoprecipitation, Transfection, Negative Control, De-Phosphorylation Assay

    Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: Schematic diagram showing the relationship of calpain/NF-κB/inflammation/NVU damage after CCI in mice. Traumatic brain injury induces calcium overload, which, in turn, upregulates calpain. Calpain may downregulate IκB and activate NF-κB. NF-κB induces activation of TNF-α, iNOS, ICAM-1, and MMP-9. These inflammatory substances induce degradation of basal lamina and tight junction proteins, resulting in NVU disruption, leading to brain edema. MDL28170 could reverse those changes. NF-κB: Nuclear factor-κB; NVU: Neurovascular unit; CCI: Controlled cortical impact; IκB: Inhibitory-κB; TNF-α: Tumor necrosis factor-α; iNOS: Inducible nitric oxide synthase; ICAM-1: Intracellular adhesion molecule-1; MMP-9: Matrix metalloproteinase-9.

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Mouse Assay, Activation Assay

    MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Journal: Chinese Medical Journal

    Article Title: Protective Effects of Calpain Inhibition on Neurovascular Unit Injury through Downregulating Nuclear Factor-κB-related Inflammation during Traumatic Brain Injury in Mice

    doi: 10.4103/0366-6999.198001

    Figure Lengend Snippet: MDL28170 treatment suppresses the calpain activity in the cytosolic and mitochondrial fractions and upregulates the expression of calpastatin in the cytosolic fractions. (a and b) The bar graphs reflect the calpain activity in the cytosolic fractions and mitochondrial fractions at 6 h and 24 h. (c) Representative Western blots of calpastatin and β-actin from each group; (d) the results were quantified and are shown as the mean ± SD. * P

    Article Snippet: [ ] In brief, cytosolic and mitochondrial proteins (30 μg) were incubated with calpain reaction buffer (20 mmol/L HEPES, 1 mmol/L EDTA, 50 mmol/L NaCl, and 0.1% (v/v) 2-mercaptoethanol, containing 10 μmol/L calpain I fluorescent substrate [Calbiochem Co., La Jolla, CA, USA], pH 7.6).

    Techniques: Activity Assay, Expressing, Western Blot

    The gene expression profile of the pancreatic stem cells cultured under condition #3. To investigate the gene expression profile of HN#101 cells cultured for 2 months under culture condition #3, an RT-PCR analysis of endodermal/pancreatic progenitor cell markers was performed. Feeder, feeder cells (STO cells, negative control); PDL 50, HN#101 cells at PDL 50 under culture condition #1; ESM + Feeder, HN#101 cells in ES media (ESM) at PDL 100 under culture condition #3; DE, differentiated cells at the definitive endoderm stage derived from ES cells (positive control); GTE, differentiated cells at the gut tube endoderm stage derived from ES cells (positive control); PP, differentiated cells at the pancreatic progenitor stage derived from ES cells (positive control); Sox17, sex-determining region Y box 17; Foxa2, forkhead box A2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Hnf, hepatocyte growth factor; Pdx1, pancreatic and duodenal homeobox factor 1.

    Journal: Cell Medicine

    Article Title: Culture Conditions for Mouse Pancreatic Stem Cells

    doi: 10.3727/215517913X666495

    Figure Lengend Snippet: The gene expression profile of the pancreatic stem cells cultured under condition #3. To investigate the gene expression profile of HN#101 cells cultured for 2 months under culture condition #3, an RT-PCR analysis of endodermal/pancreatic progenitor cell markers was performed. Feeder, feeder cells (STO cells, negative control); PDL 50, HN#101 cells at PDL 50 under culture condition #1; ESM + Feeder, HN#101 cells in ES media (ESM) at PDL 100 under culture condition #3; DE, differentiated cells at the definitive endoderm stage derived from ES cells (positive control); GTE, differentiated cells at the gut tube endoderm stage derived from ES cells (positive control); PP, differentiated cells at the pancreatic progenitor stage derived from ES cells (positive control); Sox17, sex-determining region Y box 17; Foxa2, forkhead box A2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Hnf, hepatocyte growth factor; Pdx1, pancreatic and duodenal homeobox factor 1.

    Article Snippet: #SO5094S1560), 2) complete embryonic stem (ES) cell media with 15% FBS (Millipore, Billerica, MA, USA), 3) complete ES cell media with 15% FBS on feeder layers of mitomycin C (Sigma-Aldrich)-treated STO cells [Sandos inbred mice fibroblast cell line with 6-thioguanine and ouabain resistance; American Type Culture Collection (ATCC), Manassas, VA, USA] (the same as the culture conditions used for mouse ES cells) ( ).

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Negative Control, Derivative Assay, Positive Control