beta actin controls  (Millipore)


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    Structured Review

    Millipore beta actin controls
    RECK expression is tumor-specific. The average staining intensity for RECK decreased to 13% in tumor tissue compared to normal tissue, whereas EMMPRIN staining remained unchanged (A) . Mean RECK staining intensities for the tumor subtypes ± standard error of the mean are shown in (B) . In comparison to their normal counterparts, RECK values increased gradually from 6% in clear cell carcinomas (CC) over 38% in papillary carcinomas (Pap.) and 45% in chromophobe carcinomas (Chrom.) to 96% in oncocytomas (Oncocyt.). P values are given above the pairs of columns (A , B) . RECK and EMMPRIN antibodies were checked in Western blots (C) . RECK bands at 125 kDa and 90 kDa were detected in protein lysates of 8 pairs of tumor and adjacent normal renal tissue (control: <t>beta-actin).</t> These samples were also used to show the panel of EMMPRIN bands, in our blot migrating between 35 and 74 kDa (control: beta-actin).
    Beta Actin Controls, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2596 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Renal cell neoplasias: reversion-inducing cysteine-rich protein with Kazal motifs discriminates tumor subtypes, while extracellular matrix metalloproteinase inducer indicates prognosis"

    Article Title: Renal cell neoplasias: reversion-inducing cysteine-rich protein with Kazal motifs discriminates tumor subtypes, while extracellular matrix metalloproteinase inducer indicates prognosis

    Journal: Journal of Translational Medicine

    doi: 10.1186/1479-5876-11-258

    RECK expression is tumor-specific. The average staining intensity for RECK decreased to 13% in tumor tissue compared to normal tissue, whereas EMMPRIN staining remained unchanged (A) . Mean RECK staining intensities for the tumor subtypes ± standard error of the mean are shown in (B) . In comparison to their normal counterparts, RECK values increased gradually from 6% in clear cell carcinomas (CC) over 38% in papillary carcinomas (Pap.) and 45% in chromophobe carcinomas (Chrom.) to 96% in oncocytomas (Oncocyt.). P values are given above the pairs of columns (A , B) . RECK and EMMPRIN antibodies were checked in Western blots (C) . RECK bands at 125 kDa and 90 kDa were detected in protein lysates of 8 pairs of tumor and adjacent normal renal tissue (control: beta-actin). These samples were also used to show the panel of EMMPRIN bands, in our blot migrating between 35 and 74 kDa (control: beta-actin).
    Figure Legend Snippet: RECK expression is tumor-specific. The average staining intensity for RECK decreased to 13% in tumor tissue compared to normal tissue, whereas EMMPRIN staining remained unchanged (A) . Mean RECK staining intensities for the tumor subtypes ± standard error of the mean are shown in (B) . In comparison to their normal counterparts, RECK values increased gradually from 6% in clear cell carcinomas (CC) over 38% in papillary carcinomas (Pap.) and 45% in chromophobe carcinomas (Chrom.) to 96% in oncocytomas (Oncocyt.). P values are given above the pairs of columns (A , B) . RECK and EMMPRIN antibodies were checked in Western blots (C) . RECK bands at 125 kDa and 90 kDa were detected in protein lysates of 8 pairs of tumor and adjacent normal renal tissue (control: beta-actin). These samples were also used to show the panel of EMMPRIN bands, in our blot migrating between 35 and 74 kDa (control: beta-actin).

    Techniques Used: Expressing, Staining, Western Blot

    2) Product Images from "Nuclear expression of Survivin in paediatric ependymomas and choroid plexus tumours correlates with morphologic tumour grade"

    Article Title: Nuclear expression of Survivin in paediatric ependymomas and choroid plexus tumours correlates with morphologic tumour grade

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601334

    Survivin and  β -actin protein expression in normal human ependyma and cortex.
    Figure Legend Snippet: Survivin and β -actin protein expression in normal human ependyma and cortex.

    Techniques Used: Expressing

    3) Product Images from "Restraint Stress Intensifies Interstitial K+ Accumulation during Severe Hypoxia"

    Article Title: Restraint Stress Intensifies Interstitial K+ Accumulation during Severe Hypoxia

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2012.00053

    Quantification of GFAP and Kir4.1 expression . (A) Immunolabeling of the astrocytic marker GFAP revealed an increased GFAP immunoreactivity in the hippocampal CA1 subfield in sections from stressed rats. Relative optical density of the sections was determined in st. oriens and st. radiatum (bar plots on the right). In both layers, GFAP immunoreactivity was more dense in stressed as compared to control rats. The number of sections analyzed is reported (so st. oriens , sp st. pyramidale , sr st. radiatum) . (B) Immunolabeling also revealed a downregulation of Kir4.1 in stressed rats that was obvious in all layers of the CA1 subfield. (C) Western blots confirmed the decreased Kir4.1 immunoreactivity, yielding a decreased expression of Kir4.1 as compared to β-actin content in stressed rats ( n = 8 hippocampi each group).
    Figure Legend Snippet: Quantification of GFAP and Kir4.1 expression . (A) Immunolabeling of the astrocytic marker GFAP revealed an increased GFAP immunoreactivity in the hippocampal CA1 subfield in sections from stressed rats. Relative optical density of the sections was determined in st. oriens and st. radiatum (bar plots on the right). In both layers, GFAP immunoreactivity was more dense in stressed as compared to control rats. The number of sections analyzed is reported (so st. oriens , sp st. pyramidale , sr st. radiatum) . (B) Immunolabeling also revealed a downregulation of Kir4.1 in stressed rats that was obvious in all layers of the CA1 subfield. (C) Western blots confirmed the decreased Kir4.1 immunoreactivity, yielding a decreased expression of Kir4.1 as compared to β-actin content in stressed rats ( n = 8 hippocampi each group).

    Techniques Used: Expressing, Immunolabeling, Marker, Western Blot

    4) Product Images from "Differential Relevance of NF-κB and JNK in the Pathophysiology of Hemorrhage/Resususcitation-Induced Liver Injury after Chronic Ethanol Feeding"

    Article Title: Differential Relevance of NF-κB and JNK in the Pathophysiology of Hemorrhage/Resususcitation-Induced Liver Injury after Chronic Ethanol Feeding

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137875

    Two h after the end of resuscitation, liver tissue was harvested and western blot for the phosphorylated or non- phosphorylated c-JUN (Fig 6A), p65 subunit of NF-κB and β-actin was performed. Lanes 1–4: liver protein extracts from ctrl-fed mice treated with veh, lanes 5–8: ctrl-fed mice treated with D-JNKI-1, lanes 9–12: EtOH-fed mice treated with veh and lanes 13–16: EtOH-fed mice treated with D-JNKI-1. Sham operated animals underwent the surgical procedures but hemorrhagic shock with resuscitation (H/R) was not carried out. In Fig 6B, the ratio of phosphorylated c-JUN and p65 subunit of c-JUN and NF-κB, respectively, and total protein after densitometric measurements and normalization to β-actin is represented. (*: p
    Figure Legend Snippet: Two h after the end of resuscitation, liver tissue was harvested and western blot for the phosphorylated or non- phosphorylated c-JUN (Fig 6A), p65 subunit of NF-κB and β-actin was performed. Lanes 1–4: liver protein extracts from ctrl-fed mice treated with veh, lanes 5–8: ctrl-fed mice treated with D-JNKI-1, lanes 9–12: EtOH-fed mice treated with veh and lanes 13–16: EtOH-fed mice treated with D-JNKI-1. Sham operated animals underwent the surgical procedures but hemorrhagic shock with resuscitation (H/R) was not carried out. In Fig 6B, the ratio of phosphorylated c-JUN and p65 subunit of c-JUN and NF-κB, respectively, and total protein after densitometric measurements and normalization to β-actin is represented. (*: p

    Techniques Used: Western Blot, Mouse Assay

    5) Product Images from "Tissue-specific deregulation of selected HDACs characterizes ALS progression in mouse models: pharmacological characterization of SIRT1 and SIRT2 pathways"

    Article Title: Tissue-specific deregulation of selected HDACs characterizes ALS progression in mouse models: pharmacological characterization of SIRT1 and SIRT2 pathways

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.247

    Protein expression patterns of HDAC5, HDAC11, SIRT1 and SIRT2 in differentiated human SH-SY5Y neuroblastoma cells. SH-SY5Y cells were uninfected (Ctrl) or infected with adenoviral vectors coding for wild-type SOD1 (Wt) or G93A-SOD1 (G93A). ( a ) Western blot analysis of 20 μ g of cell lysate using antibodies against HDAC5, HDAC11, SIRT1, SIRT2, p53-Ac and Ac-tubulin. β -Actin was used as loading control, SOD1 as infection control, and P -53 and tubulin to monitor the acetylation rate. ( b ) Densitometric analysis of n =3 experiments as in ( a ). Values significantly different from relative controls are indicated with ** ,## P
    Figure Legend Snippet: Protein expression patterns of HDAC5, HDAC11, SIRT1 and SIRT2 in differentiated human SH-SY5Y neuroblastoma cells. SH-SY5Y cells were uninfected (Ctrl) or infected with adenoviral vectors coding for wild-type SOD1 (Wt) or G93A-SOD1 (G93A). ( a ) Western blot analysis of 20 μ g of cell lysate using antibodies against HDAC5, HDAC11, SIRT1, SIRT2, p53-Ac and Ac-tubulin. β -Actin was used as loading control, SOD1 as infection control, and P -53 and tubulin to monitor the acetylation rate. ( b ) Densitometric analysis of n =3 experiments as in ( a ). Values significantly different from relative controls are indicated with ** ,## P

    Techniques Used: Expressing, Infection, Western Blot

    G93A-SOD1 toxicity is not mediated by p53 acetylation state or by IRS-2/Ras/ERK1/2 pathway in SH-SY5Y cells. ( a ) Western blot analysis of 20 μ M of total protein extract from cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) and treated with 3 μ M Ex527 or DMSO. Antibodies against SIRT1, Erk1/2, pErk1/2, p53, p53-Ac, tubulin and Ac-tubulin were used. β -Actin was used as loading control. One representative blot is shown from three independent experiments giving comparable results. ( b ) Densitometric analysis of results as in ( a ); data are expressed as acetylation ratio of p53 and tubulin. ( c ) Cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) were treated with 3 μ M Ex527 or with 3 μ M SL327 or both. Cell viability was assessed and reported for Figure 5a . Values significantly different from relative controls are indicated with * P
    Figure Legend Snippet: G93A-SOD1 toxicity is not mediated by p53 acetylation state or by IRS-2/Ras/ERK1/2 pathway in SH-SY5Y cells. ( a ) Western blot analysis of 20 μ M of total protein extract from cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) and treated with 3 μ M Ex527 or DMSO. Antibodies against SIRT1, Erk1/2, pErk1/2, p53, p53-Ac, tubulin and Ac-tubulin were used. β -Actin was used as loading control. One representative blot is shown from three independent experiments giving comparable results. ( b ) Densitometric analysis of results as in ( a ); data are expressed as acetylation ratio of p53 and tubulin. ( c ) Cells infected with adenoviral vectors coding for Wt-SOD1 (Wt) and G93A-SOD1 (G93A) were treated with 3 μ M Ex527 or with 3 μ M SL327 or both. Cell viability was assessed and reported for Figure 5a . Values significantly different from relative controls are indicated with * P

    Techniques Used: Western Blot, Infection

    6) Product Images from "Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells"

    Article Title: Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells

    Journal: Histochemistry and Cell Biology

    doi: 10.1007/s00418-014-1199-9

    Subcellular distribution of β- ( a ) and γ-actin ( b ) in examined cells overexpressing actin isoforms. Lower rows in a and b shows representative BE cells overexpressing β- or γ-actin, respectively. Left panel AcGFP fluorescence ( green ), middle panel endogenous β- or γ-actin stained with mouse anti-β- or anti-γ-actin antibody ( red ). Merged images are shown in the right panel . Long arrows show colocalization of AcGFP-actin and endogenous actin in lamellipodia and short ones probably in retracting tail areas. Scale bar 10 μm
    Figure Legend Snippet: Subcellular distribution of β- ( a ) and γ-actin ( b ) in examined cells overexpressing actin isoforms. Lower rows in a and b shows representative BE cells overexpressing β- or γ-actin, respectively. Left panel AcGFP fluorescence ( green ), middle panel endogenous β- or γ-actin stained with mouse anti-β- or anti-γ-actin antibody ( red ). Merged images are shown in the right panel . Long arrows show colocalization of AcGFP-actin and endogenous actin in lamellipodia and short ones probably in retracting tail areas. Scale bar 10 μm

    Techniques Used: Fluorescence, Staining

    Western blot analysis of AcGFP, β-actin and γ-actin. A representative immunoblots identificating AcGFP and fusion proteins ( a ) as well as β-actin ( b ) and γ-actin ( c ) in cellular extracts of control cells (transfected with pAcGFP-C1) and cells overexpressing AcGFP tagged β- or γ-actin. Used antibodies: mouse monoclonal antibodies directed against β tubulin, mouse monoclonal antibodies directed against GFP, mouse monoclonal anti-β-actin antibodies and mouse monoclonal anti-γ-actin antibodies
    Figure Legend Snippet: Western blot analysis of AcGFP, β-actin and γ-actin. A representative immunoblots identificating AcGFP and fusion proteins ( a ) as well as β-actin ( b ) and γ-actin ( c ) in cellular extracts of control cells (transfected with pAcGFP-C1) and cells overexpressing AcGFP tagged β- or γ-actin. Used antibodies: mouse monoclonal antibodies directed against β tubulin, mouse monoclonal antibodies directed against GFP, mouse monoclonal anti-β-actin antibodies and mouse monoclonal anti-γ-actin antibodies

    Techniques Used: Western Blot, Transfection

    7) Product Images from "Adhesion and Proliferation of Human Periodontal Ligament Cells on Poly(2-methoxyethyl acrylate)"

    Article Title: Adhesion and Proliferation of Human Periodontal Ligament Cells on Poly(2-methoxyethyl acrylate)

    Journal: BioMed Research International

    doi: 10.1155/2014/102648

    Quantification of adherent PDL cell morphologies for 1 h and 1 day. (a) CLSM images for the quantification of adherent PDL cell morphology on polymer surfaces. Scale bars: 100 μ m. Blue: nucleus, green: actin, and red: vinculin. Polymers: PET, PMEA, and PHEMA. (b) Projected cell area. (c) Perimeter of adherent PDL cells. (d) Long and short axes of adherent PDL cells. Polymers: PET, PMEA, and PHEMA. ** P
    Figure Legend Snippet: Quantification of adherent PDL cell morphologies for 1 h and 1 day. (a) CLSM images for the quantification of adherent PDL cell morphology on polymer surfaces. Scale bars: 100 μ m. Blue: nucleus, green: actin, and red: vinculin. Polymers: PET, PMEA, and PHEMA. (b) Projected cell area. (c) Perimeter of adherent PDL cells. (d) Long and short axes of adherent PDL cells. Polymers: PET, PMEA, and PHEMA. ** P

    Techniques Used: Confocal Laser Scanning Microscopy, Positron Emission Tomography

    CLSM images of PDL cells cultured on polymer surfaces. Scale bars: 300 μ m. Blue: nucleus, green: actin, and red: vinculin. Time points are 1 h, 1 day, 3 days, and 7 days. Polymers: PET, PMEA, PHEMA, and PMPC.
    Figure Legend Snippet: CLSM images of PDL cells cultured on polymer surfaces. Scale bars: 300 μ m. Blue: nucleus, green: actin, and red: vinculin. Time points are 1 h, 1 day, 3 days, and 7 days. Polymers: PET, PMEA, PHEMA, and PMPC.

    Techniques Used: Confocal Laser Scanning Microscopy, Cell Culture, Positron Emission Tomography

    Localization of nucleus, actin, and vinculin in adherent PDL cells on polymer surfaces. Scale bars: 10 μ m. In the cross-section images, the top panel shows the nucleus (blue), the second panel shows the nucleus and actin (green), the third panel shows the nucleus and vinculin (red), and the bottom panel shows a merged image. The time points are 1 h, 4 h, 1 day, and 3 days. (a) PDL cells on PET. (b) PDL cells on PMEA. (c) PDL cells on PHEMA. White arrows indicate focal adhesions that were localized at the basal cell surface. Yellow arrows indicate nonfocal adhesions (non-FA) localized at the apical cell surface. White arrowheads indicate vinculin rods that were connected vertically and penetrated the adherent cell. White circles indicate vinculin fibers that were mainly oriented along the long axis of the adherent cells. (d) Schematic representation of vinculin fiber formation.
    Figure Legend Snippet: Localization of nucleus, actin, and vinculin in adherent PDL cells on polymer surfaces. Scale bars: 10 μ m. In the cross-section images, the top panel shows the nucleus (blue), the second panel shows the nucleus and actin (green), the third panel shows the nucleus and vinculin (red), and the bottom panel shows a merged image. The time points are 1 h, 4 h, 1 day, and 3 days. (a) PDL cells on PET. (b) PDL cells on PMEA. (c) PDL cells on PHEMA. White arrows indicate focal adhesions that were localized at the basal cell surface. Yellow arrows indicate nonfocal adhesions (non-FA) localized at the apical cell surface. White arrowheads indicate vinculin rods that were connected vertically and penetrated the adherent cell. White circles indicate vinculin fibers that were mainly oriented along the long axis of the adherent cells. (d) Schematic representation of vinculin fiber formation.

    Techniques Used: Positron Emission Tomography

    8) Product Images from "Heparanase 2 attenuates head and neck tumor vascularity and growth"

    Article Title: Heparanase 2 attenuates head and neck tumor vascularity and growth

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-15-1975

    Hpa2 induces LOX expression and LOXL2 nuclear localization. A . Masson’s/Trichrome and immunostaining. Five micron sections from tumor xenografts produced by the indicated control (#3, #5) and Hpa2 over expressing (#60, #64) cell clones were stained
    Figure Legend Snippet: Hpa2 induces LOX expression and LOXL2 nuclear localization. A . Masson’s/Trichrome and immunostaining. Five micron sections from tumor xenografts produced by the indicated control (#3, #5) and Hpa2 over expressing (#60, #64) cell clones were stained

    Techniques Used: Expressing, Immunostaining, Produced, Clone Assay, Staining

    9) Product Images from "Hypoxia Inducible Factor (HIF)-1 Coordinates Induction of Toll-Like Receptors TLR2 and TLR6 during Hypoxia"

    Article Title: Hypoxia Inducible Factor (HIF)-1 Coordinates Induction of Toll-Like Receptors TLR2 and TLR6 during Hypoxia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001364

    TLR6 transcript, protein and function during hypoxia. A and B , Quantification of TLR6 transcripit levels in freshly purified blood DCs, MM6 cells, confluent HMEC-1 monolayers and confluent Caco-2 monolayers. Cells were exposed to normoxia and hypoxia for indicated time points. Total RNA was isolated and TLR6 mRNA levels were determined by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM, where transcript levels in normoxic cells were normalized to 1. Results are derived from three different experiments. *, significant differences from normoxic cells (p
    Figure Legend Snippet: TLR6 transcript, protein and function during hypoxia. A and B , Quantification of TLR6 transcripit levels in freshly purified blood DCs, MM6 cells, confluent HMEC-1 monolayers and confluent Caco-2 monolayers. Cells were exposed to normoxia and hypoxia for indicated time points. Total RNA was isolated and TLR6 mRNA levels were determined by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM, where transcript levels in normoxic cells were normalized to 1. Results are derived from three different experiments. *, significant differences from normoxic cells (p

    Techniques Used: Purification, Isolation, Quantitative RT-PCR, Derivative Assay

    Expression of TLR2 and TLR6 during ambient hypoxia in vivo. A and B , Expression of TLR2 and TLR6 mRNA in normoxic or hypoxic organs. Tissue of Colon, Liver and Lung where harvested from mice after exposure to normoxia or normobaric hypoxia (8% O2, 92% N2 for 6h). Total RNA was isolated, and quantitative mRNA levels of TLR2 and TLR6 were assessed by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM and transcript levels in normoxic organs were normalized to 1. Results are derived from six animals in each condition (*p
    Figure Legend Snippet: Expression of TLR2 and TLR6 during ambient hypoxia in vivo. A and B , Expression of TLR2 and TLR6 mRNA in normoxic or hypoxic organs. Tissue of Colon, Liver and Lung where harvested from mice after exposure to normoxia or normobaric hypoxia (8% O2, 92% N2 for 6h). Total RNA was isolated, and quantitative mRNA levels of TLR2 and TLR6 were assessed by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM and transcript levels in normoxic organs were normalized to 1. Results are derived from six animals in each condition (*p

    Techniques Used: Expressing, In Vivo, Mouse Assay, Isolation, Quantitative RT-PCR, Derivative Assay

    Influence of hypoxia on TLR mRNA expression in murine dendritic cells. Isolated murine bone-marrow-derived dendritic cells (BMDCs) were exposed to normoxia or hypoxia for 24 hours. Total RNA was isolated, and quantitative mRNA levels of TLR1-9 and TLR11-13 were assessed by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM and transcript levels in normoxic BMDCs were normalized to 1. Results are derived from three different experiments (*p
    Figure Legend Snippet: Influence of hypoxia on TLR mRNA expression in murine dendritic cells. Isolated murine bone-marrow-derived dendritic cells (BMDCs) were exposed to normoxia or hypoxia for 24 hours. Total RNA was isolated, and quantitative mRNA levels of TLR1-9 and TLR11-13 were assessed by real-time RT-PCR. Data were calculated relative to ß-actin and expressed as fold change relative to normoxia±SEM and transcript levels in normoxic BMDCs were normalized to 1. Results are derived from three different experiments (*p

    Techniques Used: Expressing, Isolation, Derivative Assay, Quantitative RT-PCR

    Influence of hypoxia inducible factor (HIF)-1α on TLR6 expression during hypoxia. A , Map of TLR6 promoter region showing positions of the putative HIF binding sites and the binding site for NFκB relative to the transcription start site (TSS). B , Stable transfected HMEC-1 monolayers containing either HIF-1α siRNA or control-siRNA were exposed to normoxia or hypoxia for indicated time points. Total RNA was isolated, and 1 µg of RNA was transcribed into first strand cDNA. Relative expressional levels of TLR6 transcripts were compared to normoxic controls by real-time RT-PCR. Data were calculated relative to internal control gene (ß-actin), and are expressed as fold change over normoxia±SEM, *, significant differences from normoxia and control cells. Results are derived from three different experiments in each condition. C , Total RNA of normoxic monolayers of either wildtype (WT) or oxygen-stable HIF-1α expressing (HIF +/+ ) HMEC-1 cells was isolated and realt-time RT-PCR was performed as described above. *, significant differences from wildtype cells. D , Western blot analysis of TLR6 protein of normoxic HMEC-1 wildtype (WT) and oxygen-stable HIF-1α expressing (HIF+/+) cells. The same blot was probed for ß-actin expression as a control for protein loading. E , HMEC-1 monolayers were treated with 1mM of dimethyloxalylglycine (DMOG) for 24 hours. Afterwards transcript levels of TLR6 where quantified by real-time RT-PCR as described above. *, significant differences from untreated cells. F , ChIP assay was utilized to examine HIF-1α binding to the TLR6 promoter in normoxic and hypoxic HMEC-1 cells. Reaction controls included immunoprecipitations using a nonspecific igG monoclonal antibody (IgG) and PCR performed using HMEC-1 DNA (input). An example of three experiments is shown.
    Figure Legend Snippet: Influence of hypoxia inducible factor (HIF)-1α on TLR6 expression during hypoxia. A , Map of TLR6 promoter region showing positions of the putative HIF binding sites and the binding site for NFκB relative to the transcription start site (TSS). B , Stable transfected HMEC-1 monolayers containing either HIF-1α siRNA or control-siRNA were exposed to normoxia or hypoxia for indicated time points. Total RNA was isolated, and 1 µg of RNA was transcribed into first strand cDNA. Relative expressional levels of TLR6 transcripts were compared to normoxic controls by real-time RT-PCR. Data were calculated relative to internal control gene (ß-actin), and are expressed as fold change over normoxia±SEM, *, significant differences from normoxia and control cells. Results are derived from three different experiments in each condition. C , Total RNA of normoxic monolayers of either wildtype (WT) or oxygen-stable HIF-1α expressing (HIF +/+ ) HMEC-1 cells was isolated and realt-time RT-PCR was performed as described above. *, significant differences from wildtype cells. D , Western blot analysis of TLR6 protein of normoxic HMEC-1 wildtype (WT) and oxygen-stable HIF-1α expressing (HIF+/+) cells. The same blot was probed for ß-actin expression as a control for protein loading. E , HMEC-1 monolayers were treated with 1mM of dimethyloxalylglycine (DMOG) for 24 hours. Afterwards transcript levels of TLR6 where quantified by real-time RT-PCR as described above. *, significant differences from untreated cells. F , ChIP assay was utilized to examine HIF-1α binding to the TLR6 promoter in normoxic and hypoxic HMEC-1 cells. Reaction controls included immunoprecipitations using a nonspecific igG monoclonal antibody (IgG) and PCR performed using HMEC-1 DNA (input). An example of three experiments is shown.

    Techniques Used: Expressing, Binding Assay, Transfection, Isolation, Quantitative RT-PCR, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    Role of hypoxia inducible factor (HIF)-1 in TLR2 and TLR6 expression during hypoxia in vivo. Real-time RT-PCR analysis of murine epithelial TLR2 and TLR6 mRNA in conditional HIF-1α mutant (HIF-/-) and littermate control (WT) animals subjected to normoxia or hypoxia. Data were calculated relative to ß-actin and are expressed as fold change over normoxia±SEM, where transcript levels of control animals were normalized to 1. *, significant differences from normoxic control animals (p
    Figure Legend Snippet: Role of hypoxia inducible factor (HIF)-1 in TLR2 and TLR6 expression during hypoxia in vivo. Real-time RT-PCR analysis of murine epithelial TLR2 and TLR6 mRNA in conditional HIF-1α mutant (HIF-/-) and littermate control (WT) animals subjected to normoxia or hypoxia. Data were calculated relative to ß-actin and are expressed as fold change over normoxia±SEM, where transcript levels of control animals were normalized to 1. *, significant differences from normoxic control animals (p

    Techniques Used: Expressing, In Vivo, Quantitative RT-PCR, Mutagenesis

    10) Product Images from "Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) *Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) * ♦"

    Article Title: Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) *Seven in Absentia Homolog 2 (Siah2) Protein Is a Regulator of NF-E2-related Factor 2 (Nrf2) * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.438762

    Effect of hypoxia on Nrf2 accumulation and transcription. A, Hep3B, HEK293, and HeLa cells were exposed to hypoxia for 3, 6, or 9 h. Twenty, 40, and 5 μg of Hep3B lysates prepared were immunoblotted with anti-Nrf2, anti-HIF-1α, or anti-β-actin
    Figure Legend Snippet: Effect of hypoxia on Nrf2 accumulation and transcription. A, Hep3B, HEK293, and HeLa cells were exposed to hypoxia for 3, 6, or 9 h. Twenty, 40, and 5 μg of Hep3B lysates prepared were immunoblotted with anti-Nrf2, anti-HIF-1α, or anti-β-actin

    Techniques Used:

    Effect of hypoxia on Siah2 accumulation. Hep3B cells were exposed to hypoxia for 6 h. Sixty, 40, 20, and 5 μg of lysates were immunoblotted with anti-Siah2, anti-HIF-1α, anti-Nrf2, or anti-β-actin antibodies.
    Figure Legend Snippet: Effect of hypoxia on Siah2 accumulation. Hep3B cells were exposed to hypoxia for 6 h. Sixty, 40, 20, and 5 μg of lysates were immunoblotted with anti-Siah2, anti-HIF-1α, anti-Nrf2, or anti-β-actin antibodies.

    Techniques Used:

    11) Product Images from "ORP150/HSP12A Regulates Purkinje Cell Survival: A Role for Endoplasmic Reticulum Stress in Cerebellar Development"

    Article Title: ORP150/HSP12A Regulates Purkinje Cell Survival: A Role for Endoplasmic Reticulum Stress in Cerebellar Development

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4029-03.2004

    Expression of ORP150 in cerebellum of genetically manipulated mice. Immunoblotting was performed on protein extracts from ORP150 +/- mice ( A ), non-Tg littermates (ORP150 +/+ ; H ) or Tg ORP150 mice ( P ) at postnatal days 0-30 using antibody to ORP150 (top lanes) or β-actin (bottom lanes). In each panel, densitometric analysis of ORP150 band intensity was performed, and statistical analysis of four representative experiments is shown. Values are expressed as fold increase versus antigen level of ORP150 in the cerebellum in non-Tg littermates at the time of birth; n = 6 per time point. ** p
    Figure Legend Snippet: Expression of ORP150 in cerebellum of genetically manipulated mice. Immunoblotting was performed on protein extracts from ORP150 +/- mice ( A ), non-Tg littermates (ORP150 +/+ ; H ) or Tg ORP150 mice ( P ) at postnatal days 0-30 using antibody to ORP150 (top lanes) or β-actin (bottom lanes). In each panel, densitometric analysis of ORP150 band intensity was performed, and statistical analysis of four representative experiments is shown. Values are expressed as fold increase versus antigen level of ORP150 in the cerebellum in non-Tg littermates at the time of birth; n = 6 per time point. ** p

    Techniques Used: Expressing, Mouse Assay

    Expression of ORP150 in brain during postnatal development. A-D , Protein extracts prepared from cerebral cortex (CCx; A ), caudate putamen (Cpu; B ), hippocampus (Hip; C ), or cerebellum (Cb; D ) during development (0-20 d after birth) were subjected to Western blotting using antibody to ORP150 (top lanes), GRP78 (middle lanes), or β-actin (bottom lanes). In each blot, densitometric analysis of the ORP150 band intensity was performed and expressed by fold increase versus that on day 0; n = 6 per time point. ** p
    Figure Legend Snippet: Expression of ORP150 in brain during postnatal development. A-D , Protein extracts prepared from cerebral cortex (CCx; A ), caudate putamen (Cpu; B ), hippocampus (Hip; C ), or cerebellum (Cb; D ) during development (0-20 d after birth) were subjected to Western blotting using antibody to ORP150 (top lanes), GRP78 (middle lanes), or β-actin (bottom lanes). In each blot, densitometric analysis of the ORP150 band intensity was performed and expressed by fold increase versus that on day 0; n = 6 per time point. ** p

    Techniques Used: Expressing, Western Blot

    12) Product Images from "SPARCL1 suppresses metastasis in prostate cancer"

    Article Title: SPARCL1 suppresses metastasis in prostate cancer

    Journal: Molecular Oncology

    doi: 10.1016/j.molonc.2013.07.008

    in vitro evaluation of SPARCL1 function. (A) Western Blot was used to confirm SPARCL1 expression in PC3‐luc/SPARCL1 in comparison to control PC3‐luc/EV cells. The Western blot membrane was re‐probed with anti‐β‐actin.
    Figure Legend Snippet: in vitro evaluation of SPARCL1 function. (A) Western Blot was used to confirm SPARCL1 expression in PC3‐luc/SPARCL1 in comparison to control PC3‐luc/EV cells. The Western blot membrane was re‐probed with anti‐β‐actin.

    Techniques Used: In Vitro, Western Blot, Expressing

    13) Product Images from "Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide"

    Article Title: Blimp1 Activation by AP-1 in Human Lung Cancer Cells Promotes a Migratory Phenotype and Is Inhibited by the Lysyl Oxidase Propeptide

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033287

    Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0). P values were calculated using a Student's t -test. *, P
    Figure Legend Snippet: Blimp1 promotes lung cancer cell migration and is aberrantly expressed in multiple cancers. (A) A549 cells or (B) H441 cells were transiently transfected with 1 µg of Blimp1 cDNA or EV DNA using Lipofectamine 2000. Upper panels: WCE were isolated after 48 h and subjected to immunoblot analysis for Blimp1 and β-actin. Lower panels: Alternatively, 24 h after transfection, cells were subjected to a migration assay as in Fig. 1 . The average migration from three independent experiments ± SD is presented relative to the EV (set at 1.0). P values were calculated using a Student's t -test. *, P

    Techniques Used: Migration, Transfection, Isolation

    A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).
    Figure Legend Snippet: A Ras to c-Raf pathway induces the Blimp1 promoter and AP-1 activity. (A) A549 cells were transfected with 5 µg of a plasmid expressing dominant negative Ras S186 or EV DNA. After 48 h, WCE and RNA were prepared. Samples (30 µg) of WCE were subjected to immunoblot analysis for Blimp1, Ras and α-tubulin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. The average values for normalized Blimp1 levels from two independent experiments are given relative to EV DNA (set to 1.0). (B) RNA was isolated from the A549 cells treated as in part A, and subjected to Q-PCR for BLIMP1 mRNA and normalized to GAPDH . The values represent an average of two independent experiments. (C) A549 cells were transfected, in triplicate, with 0.16 µg of Ras S186 plasmid or EV DNA, 0.33 µg of a MSV- β-gal expression vector and 0.16 µg of the 7-kB Blimp1 promoter Blimp1 -Luc, in a 12-well plate. After 48 h, cell lysates were subjected to measurements for luciferase and β-gal activities and normalized Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0). (D) Two-hundred pmol of an siRNA against K-Ras or a negative control siRNA (Ctrl) was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for K-Ras, Blimp1, c-Jun, phospho-ERK (p-ERK), Fra-1, Fra-2, and α-tubulin. Average normalized levels of Blimp1, c-Jun, Fra-1, Fra-2 and K-Ras from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (E) Two-hundred pmol of an siRNA against c- RAF or a negative control siRNA was incubated in the presence of 25 µl of Lipofectamine RNAiMAX in 2 ml of optiMEM in P100 plates. A549 cells (6.4×10 5 ) were seeded at a final siRNA concentration of 20 nM for 48 h. WCE were subjected to immunoblotting for c-Raf, Blimp1, Fra-1, Fra-2, c-Jun, and α-tubulin. Average normalized levels of c-Raf, Blimp1, Fra-1, Fra-2 and c-Jun from two independent experiments are given relative to the control (set to 1.0). Immunoblots from one of two independent experiments with similar results are presented. (F) A549 cells were transiently transfected, in triplicate, with si-c-RAF or negative control siRNA at a final concentration of 20 nM in a 12-well plate. Eight h later, Blimp1 -luc promoter construct (0.16 µg) and an MSV- β-gal expression vector (0.33 µg) were transfected into these siRNA-treated A549 cells for an additional 40 h. Relative (Rel.) Blimp1 promoter activity values are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Dominant Negative Mutation, Software, Isolation, Polymerase Chain Reaction, Luciferase, Negative Control, Incubation, Concentration Assay, Western Blot, Construct

    Blimp1 is expressed in lung cancer cells and its knockdown reduces migration. (A) Samples of nuclear extracts (20 µg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were subjected to immunoblotting for Blimp1 and β-actin, as a control for equal loading. Positions of molecular weight markers are given in the left lane. A representative of two independent experiments with similar results is shown. (B) A549 and (C) H1299 cells were transiently transfected with 10 nM each of siBLIMP1-1 , siBLIMP1-2 or a scrambled negative control siRNA. Upper panels: Forty-eight h after transfection, WCE (30 µg) were subjected to immunoblotting for Blimp1 and β-actin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. Normalized Blimp1 expression was determined in two independent experiments and the average values are given below the blots. Lower panels: Alternatively, after 24 h, cultures were trypsinized and 1×10 5 cells subjected to a migration assay for 16 h, in triplicate. The average migration from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). P values were calculated using Student's t -test. *, P
    Figure Legend Snippet: Blimp1 is expressed in lung cancer cells and its knockdown reduces migration. (A) Samples of nuclear extracts (20 µg) of A549, H1299, Calu-1, H23 and H441 human lung cancer cells and MCF-7 and MDA-MB-231 (MB-231) breast cancer cells were subjected to immunoblotting for Blimp1 and β-actin, as a control for equal loading. Positions of molecular weight markers are given in the left lane. A representative of two independent experiments with similar results is shown. (B) A549 and (C) H1299 cells were transiently transfected with 10 nM each of siBLIMP1-1 , siBLIMP1-2 or a scrambled negative control siRNA. Upper panels: Forty-eight h after transfection, WCE (30 µg) were subjected to immunoblotting for Blimp1 and β-actin. The bands were quantified using NIH Image J software and Blimp1 expression normalized to β-actin expression. Normalized Blimp1 expression was determined in two independent experiments and the average values are given below the blots. Lower panels: Alternatively, after 24 h, cultures were trypsinized and 1×10 5 cells subjected to a migration assay for 16 h, in triplicate. The average migration from three independent experiments ± SD is presented relative to the negative control siRNA (set at 1.0). P values were calculated using Student's t -test. *, P

    Techniques Used: Migration, Multiple Displacement Amplification, Molecular Weight, Transfection, Negative Control, Software, Expressing

    Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.
    Figure Legend Snippet: Ectopic LOX-PP reduces Blimp1 expression in lung cancer cells. (A) H1299-EV cells, and H1299-LOX-PP4 (PP4) and H1299-LOX-PP7 (PP7) clones, isolated as described previously [25] , were treated in triplicate with 2 µg/ml dox for 48 h. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). (B) A549-EV, A549-hLOX-PP, A549-mLOX-PP dox-inducible stable populations were treated with 2 µg/ml dox for 48 h in DMEM supplemented with 0.5% FBS. FBS was added back to 10% and cells incubated overnight. RNA from two independent experiments was subjected to Q-PCR and normalized values for BLIMP1 mRNA relative to GAPDH levels are presented as the mean ± SEM (EV DNA set to 1.0). Samples of medium (5 ml) were subjected to immunoprecipitation followed by immunoblotting using V5 antibody for LOX-PP expression. (C) A549 and H1299 cells were transiently transfected with human LOX-PP cDNA or EV DNA. After 48 h, media and WCE were prepared. Samples of media (50 µl) were subjected to immunoblotting for V5. Samples of WCE (25 µg) were probed for Blimp1 and β-actin, and average normalized Blimp1 values from two independent experiments presented relative to EV DNA, set to 1.0. (D) A549 and H441 cells were treated with purified recombinant LOX-PP protein at a final concentration of 4 or 1 µg/ml, respectively, or the same volume of vehicle (water) in medium with 0.5% FBS. Twenty-four h later, FBS was added back to 10% and cultures incubated overnight. WCE were subjected to immunoblotting for Blimp1, phospho-c-Jun (p-c-Jun), total c-Jun, Fra-1 and Fra-2 and α-tubulin, as a loading control. Normalized Blimp1 and AP-1 subunit values from two independent experiments are presented relative to EV DNA, set to 1.0.

    Techniques Used: Expressing, Isolation, Polymerase Chain Reaction, Incubation, Immunoprecipitation, Transfection, Purification, Recombinant, Concentration Assay

    Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).
    Figure Legend Snippet: Ectopic AP-1 subunits induce Blimp1 expression. (A) H441 cells, growing in 6-well plates, were transfected with 1 µg of vectors expressing the indicated AP-1 subunits or EV DNA (see bottom) to make a 2 µg total. Upper panel. After 48 h, RNA was isolated and subjected to Q-PCR. The levels of BLIMP1 mRNA normalized to GAPDH mRNA are presented as mean ± SD of three independent experiments. Middle and lower panels. WCE were isolated and subjected to immunoblotting (IB) for Blimp1 (Middle panels), and for c-Jun, Fra-1, Fra-2, c-Fos and β-actin (Lower panels). (L exp., longer exposure; S exp., shorter exposure). Blimp1 levels, normalized to β-actin, were determined as in Fig. 1C and average values from two independent experiments presented relative to EV DNA, set to 1.0. (B) H441 cells were transiently transfected, in triplicate, with 0.3 µg of Blimp1 -Luc, 0.3 µg of MSV-β-gal, and vectors expressing the indicated AP-1 subunits (0.15 µg each) and EV DNA to a total of 1.0 µg DNA. Normalized values of Blimp1 promoter activity are presented as the mean ± SEM from two experiments (EV DNA set to 1.0).

    Techniques Used: Expressing, Transfection, Isolation, Polymerase Chain Reaction, Activity Assay

    14) Product Images from "Photodynamic therapy inhibits p-glycoprotein mediated multidrug resistance via JNK activation in human hepatocellular carcinoma using the photosensitizer pheophorbide a"

    Article Title: Photodynamic therapy inhibits p-glycoprotein mediated multidrug resistance via JNK activation in human hepatocellular carcinoma using the photosensitizer pheophorbide a

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-8-56

    Pa-PDT activates JNK-mediated apoptosis in R-HepG2 cells . (A) Changes in apoptosis regulatory proteins in Pa-PDT treated R-HepG2 cells. Cells (3 × 10 6 ) were treated with solvent (0.04% ethanol (CTL)) or 0.6 μM Pa-PDT, and collected at appropriate time points, where solvent control was collected at 30 min after the treatment. The cell lysates were prepared and the changes in the level of various apoptosis-related proteins were analyzed using Western blotting. The protein expression levels were semi-quantified and shown as relative intensities normalized with the band intensity of the housekeeping β-actin in each sample. Representative results from a single experiment are shown from 5 independent experiments. (B) To determinate the effect of Pa-PDT on Δψm, flow cytometry analysis was conducted in R-HepG2 cells (3 × 10 5 /well) 1 h after light illumination (84 J/cm 2 , 20 min) with 0.6 μM Pa, 0.8 μM Pa, or 0.8 μM Pa with 0.5 μM JNK inhibitor. The cells were then stained with JC-1 (10 μM) for 15 min. The green and red fluorescence of JC-1 were acquired subsequently with a flow cytometer, and the results shown as mean ± SD of 3 independent experiments.
    Figure Legend Snippet: Pa-PDT activates JNK-mediated apoptosis in R-HepG2 cells . (A) Changes in apoptosis regulatory proteins in Pa-PDT treated R-HepG2 cells. Cells (3 × 10 6 ) were treated with solvent (0.04% ethanol (CTL)) or 0.6 μM Pa-PDT, and collected at appropriate time points, where solvent control was collected at 30 min after the treatment. The cell lysates were prepared and the changes in the level of various apoptosis-related proteins were analyzed using Western blotting. The protein expression levels were semi-quantified and shown as relative intensities normalized with the band intensity of the housekeeping β-actin in each sample. Representative results from a single experiment are shown from 5 independent experiments. (B) To determinate the effect of Pa-PDT on Δψm, flow cytometry analysis was conducted in R-HepG2 cells (3 × 10 5 /well) 1 h after light illumination (84 J/cm 2 , 20 min) with 0.6 μM Pa, 0.8 μM Pa, or 0.8 μM Pa with 0.5 μM JNK inhibitor. The cells were then stained with JC-1 (10 μM) for 15 min. The green and red fluorescence of JC-1 were acquired subsequently with a flow cytometer, and the results shown as mean ± SD of 3 independent experiments.

    Techniques Used: CTL Assay, Western Blot, Expressing, Flow Cytometry, Cytometry, Staining, Fluorescence

    Pa-PDT inhibits p-glycoprotein mediated MDR in R-HepG2 cells . (A) Differential expression of MDR proteins in Pa-PDT treated R-HepG2 cells. Cells (3 × 10 6 ) were treated with Pa alone (0.4 μM Pa without PDT) or (0.4 μM, 0.6 μM, or 0.8 μM) Pa for 2 h and then with light illumination (84 J/cm 2 ) for 20 min. Cells were collected at 2 h after PDT treatment, then cell lysates were analyzed using Western blotting. The protein expression levels were semi-quantified and shown as relative intensities normalized with the band intensity of the housekeeping β-actin in each sample. Representative results from a single experiment are shown from 5 independent experiments. (B) For the intracellular accumulation of Dox, cells (4 × 10 5 /well) were treated with 0.04% ethanol (CTL, black solid), 0.6 μM (black line) and 0.8 μM (gray line) of Pa-PDT, and then the culture medium was changed to 4 μM Dox and further incubated for 2 h at 37°C, 5% CO2. The cells were collected and the intensity of Dox fluorescence was measured by a flow cytometer. (C) For detection of P-glycoprotein activity, R-HepG2 cells (1 × 10 4 /well) were pre-incubated with 0.04% ethanol (solvent control), 0.6 μM Pa (dark control), 0.6 μM Pa-PDT, or 0.6 μM Pa-PDT with 0.5 μM JNK inhibitor in a 6-well plate for 2 h and then the samples were illuminated with PDT. The treated cells were stained with 10 μM Rh-123 for 2 h at 37°C, 5% CO2 and then incubated with 5 μg/ml PI for further 15 min at room temperature. The cells were collected and analyzed by a flow cytometer, where the lower right quadrant (Rh-123 positive and PI negative) represents the cells that have intact plasma membrane but with down-regulated P-glycoprotein activity. The figure is a representative of 5 experiments and the results shown as mean ± SD.
    Figure Legend Snippet: Pa-PDT inhibits p-glycoprotein mediated MDR in R-HepG2 cells . (A) Differential expression of MDR proteins in Pa-PDT treated R-HepG2 cells. Cells (3 × 10 6 ) were treated with Pa alone (0.4 μM Pa without PDT) or (0.4 μM, 0.6 μM, or 0.8 μM) Pa for 2 h and then with light illumination (84 J/cm 2 ) for 20 min. Cells were collected at 2 h after PDT treatment, then cell lysates were analyzed using Western blotting. The protein expression levels were semi-quantified and shown as relative intensities normalized with the band intensity of the housekeeping β-actin in each sample. Representative results from a single experiment are shown from 5 independent experiments. (B) For the intracellular accumulation of Dox, cells (4 × 10 5 /well) were treated with 0.04% ethanol (CTL, black solid), 0.6 μM (black line) and 0.8 μM (gray line) of Pa-PDT, and then the culture medium was changed to 4 μM Dox and further incubated for 2 h at 37°C, 5% CO2. The cells were collected and the intensity of Dox fluorescence was measured by a flow cytometer. (C) For detection of P-glycoprotein activity, R-HepG2 cells (1 × 10 4 /well) were pre-incubated with 0.04% ethanol (solvent control), 0.6 μM Pa (dark control), 0.6 μM Pa-PDT, or 0.6 μM Pa-PDT with 0.5 μM JNK inhibitor in a 6-well plate for 2 h and then the samples were illuminated with PDT. The treated cells were stained with 10 μM Rh-123 for 2 h at 37°C, 5% CO2 and then incubated with 5 μg/ml PI for further 15 min at room temperature. The cells were collected and analyzed by a flow cytometer, where the lower right quadrant (Rh-123 positive and PI negative) represents the cells that have intact plasma membrane but with down-regulated P-glycoprotein activity. The figure is a representative of 5 experiments and the results shown as mean ± SD.

    Techniques Used: Expressing, Western Blot, CTL Assay, Incubation, Fluorescence, Flow Cytometry, Cytometry, Activity Assay, Staining

    15) Product Images from "Ser276 Phosphorylation of NF-kB p65 by MSK1 Controls SCF Expression in Inflammation"

    Article Title: Ser276 Phosphorylation of NF-kB p65 by MSK1 Controls SCF Expression in Inflammation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004393

    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.
    Figure Legend Snippet: Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.

    Techniques Used: Expressing, Transfection, Luciferase, Construct, Incubation, Activity Assay, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    16) Product Images from "The heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase"

    Article Title: The heat shock protein amplifier arimoclomol improves refolding, maturation and lysosomal activity of glucocerebrosidase

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2018.11.037

    Arimoclomol increases the quantity and ER to golgi maturation of mutated GCase in primary GD patient fibroblasts. a) Level of GBA mRNA expression in primary GD, WT and carrier fibroblasts relative to the expression level in GM00498 (WT/WT #1) cells (stippled line). b) WB analysis of GCase protein levels in primary GD fibroblasts, carrier and WT cell lines. Vinculin was used as loading control. c) Basal level of GCase activity (Fluorescence units (FLU) normalized to cell density) in the primary GD fibroblasts, carrier and WT cell lines. Data is reported as mean + SEM of 3–4 experiments/cell line. d-f) WB analysis of GCase in (d) nGD fibroblasts, (e) non-neuronopathic GD or (f) carrier and WT cell lines treated with the indicated concentrations of arimoclomol for 5 days. Lysates were subjected to EndoH-digestion analysis and the EndoH-resistant fraction is marked by **. RPA or Vinculin served as loading control for these experiments. Quantification of GCase bands on WBs is shown as arbitrary units (A.U.) with the EndoH sensitive part of the GCase band in gray and the EndoH resistant part in black. WBs are representative of 3 independent experiments/cell line.
    Figure Legend Snippet: Arimoclomol increases the quantity and ER to golgi maturation of mutated GCase in primary GD patient fibroblasts. a) Level of GBA mRNA expression in primary GD, WT and carrier fibroblasts relative to the expression level in GM00498 (WT/WT #1) cells (stippled line). b) WB analysis of GCase protein levels in primary GD fibroblasts, carrier and WT cell lines. Vinculin was used as loading control. c) Basal level of GCase activity (Fluorescence units (FLU) normalized to cell density) in the primary GD fibroblasts, carrier and WT cell lines. Data is reported as mean + SEM of 3–4 experiments/cell line. d-f) WB analysis of GCase in (d) nGD fibroblasts, (e) non-neuronopathic GD or (f) carrier and WT cell lines treated with the indicated concentrations of arimoclomol for 5 days. Lysates were subjected to EndoH-digestion analysis and the EndoH-resistant fraction is marked by **. RPA or Vinculin served as loading control for these experiments. Quantification of GCase bands on WBs is shown as arbitrary units (A.U.) with the EndoH sensitive part of the GCase band in gray and the EndoH resistant part in black. WBs are representative of 3 independent experiments/cell line.

    Techniques Used: Expressing, Western Blot, Activity Assay, Fluorescence, Recombinase Polymerase Amplification

    17) Product Images from "P2X7 receptor antagonism modulates IL-1β and MMP9 in human atherosclerotic vessels"

    Article Title: P2X7 receptor antagonism modulates IL-1β and MMP9 in human atherosclerotic vessels

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-05137-y

    Ex-vivo vessels culture at t1: A740003 effects on IL1β. IL-1β gene expression at t1 in PLs (n = 15) and IMA (n = 3) treated with A740003 ( A ) is shown. Red dotted line indicates ctrl value. Representative cropped western blot shows P2X7 and pro-IL1β expression in PLs protein extracts ( B ). Representative confocal microscopy 2D free projection max images from PLs intima are in ( C and D ). In ( C ) P2X7 (green), IL-1β (red) and caspase-1 (green) are shown. In D upper row CD68 (red), IL-1β (green) and sm22 (white) signals are merged. In ( D ) 2 nd row is IL-1β and a white square indicates the regions magnified (3x zoom) in ( D ) 3–5 th rows. Nuclei are stained with DAPI (blue). IL-1β content in extracts (n = 25) and supernatant from PL (n = 23) untreated/treated with A740003 ( E ) is displayed. Values are shown as boxes with 5–95 percentile (• indicates outlier). Paired t-test was used. Significant difference is shown as **p
    Figure Legend Snippet: Ex-vivo vessels culture at t1: A740003 effects on IL1β. IL-1β gene expression at t1 in PLs (n = 15) and IMA (n = 3) treated with A740003 ( A ) is shown. Red dotted line indicates ctrl value. Representative cropped western blot shows P2X7 and pro-IL1β expression in PLs protein extracts ( B ). Representative confocal microscopy 2D free projection max images from PLs intima are in ( C and D ). In ( C ) P2X7 (green), IL-1β (red) and caspase-1 (green) are shown. In D upper row CD68 (red), IL-1β (green) and sm22 (white) signals are merged. In ( D ) 2 nd row is IL-1β and a white square indicates the regions magnified (3x zoom) in ( D ) 3–5 th rows. Nuclei are stained with DAPI (blue). IL-1β content in extracts (n = 25) and supernatant from PL (n = 23) untreated/treated with A740003 ( E ) is displayed. Values are shown as boxes with 5–95 percentile (• indicates outlier). Paired t-test was used. Significant difference is shown as **p

    Techniques Used: Ex Vivo, Expressing, Western Blot, Confocal Microscopy, Staining

    18) Product Images from "Neuronal and Nonneuronal Cholinergic Structures in the Mouse Gastrointestinal Tract and Spleen"

    Article Title: Neuronal and Nonneuronal Cholinergic Structures in the Mouse Gastrointestinal Tract and Spleen

    Journal: The Journal of comparative neurology

    doi: 10.1002/cne.23376

    Western blot analysis and immunohistochemistry for tdTomato. A: Immunoblot for tdTomato using a commercially available anti-DsRed antibody, showing a band of 58 kDa (green). The other band corresponds to β-actin (red). Samples were obtained from
    Figure Legend Snippet: Western blot analysis and immunohistochemistry for tdTomato. A: Immunoblot for tdTomato using a commercially available anti-DsRed antibody, showing a band of 58 kDa (green). The other band corresponds to β-actin (red). Samples were obtained from

    Techniques Used: Western Blot, Immunohistochemistry

    19) Product Images from "Activated PAK4 Regulates Cell Adhesion and Anchorage-Independent Growth"

    Article Title: Activated PAK4 Regulates Cell Adhesion and Anchorage-Independent Growth

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.10.3523-3533.2001

    Morphological changes in fibroblasts stably expressing PAK4(S445N). (A) Representative Western blot analysis of Myc-tagged PAK4 expression in the different stable cell lines. Cell lysates (25 μg) from Rat1 cells or NIH 3T3 cells stably expressing the indicated plasmids were used in each blot. Blots were probed with anti-Myc antibody (top and middle panels) or anti-PAK4 antibody (bottom panel). (B) Rat1 cells expressing PAK4(S445N) produce filopodia after plating onto fibronectin. Cells expressing empty vector (pLPC) or PAK4(S445N) were plated onto fibronectin-coated coverslips and visualized by Zeiss Axiovert phase-contrast microscopy 10 min and 1 h after plating under a 100× objective. Cell morphology was visualized by time lapse photography and individual frames are shown in the figure. Panels i, ii, and iii show a PAK4(S445N) cell that was photographed at 10-s intervals 10 min after plating. Panel iv shows an example of a PAK4(S445N) cell that had already begun to spread onto the fibronectin-coated surface 1 h after plating. Panels v and vi show control cells 10 min and 1 h, respectively, after plating. (C) Morphological changes in Rat1 cells overexpressing PAK(S445N). Top panels, cells containing either PAK4(S445N) or empty vector (pLPC) were visualized by phase-contrast microscopy under a 10× objective 24 h after plating; middle panels, cells were fixed 24 h after plating onto fibronectin-coated coverslips, and polymerized actin was visualized under a 60× oil lens after staining with FITC-conjugated phalloidin; bottom panels, cells were fixed 24 h after plating, and focal adhesions were visualized by immunofluorescence microscopy under a 60× oil lens after staining with anti-vinculin antibody and rhodamine-conjugated secondary antibody. (D) Morphological changes in NIH 3T3 cells expressing PAK4(S445N). NIH 3T3 cells were analyzed as described for panel C.
    Figure Legend Snippet: Morphological changes in fibroblasts stably expressing PAK4(S445N). (A) Representative Western blot analysis of Myc-tagged PAK4 expression in the different stable cell lines. Cell lysates (25 μg) from Rat1 cells or NIH 3T3 cells stably expressing the indicated plasmids were used in each blot. Blots were probed with anti-Myc antibody (top and middle panels) or anti-PAK4 antibody (bottom panel). (B) Rat1 cells expressing PAK4(S445N) produce filopodia after plating onto fibronectin. Cells expressing empty vector (pLPC) or PAK4(S445N) were plated onto fibronectin-coated coverslips and visualized by Zeiss Axiovert phase-contrast microscopy 10 min and 1 h after plating under a 100× objective. Cell morphology was visualized by time lapse photography and individual frames are shown in the figure. Panels i, ii, and iii show a PAK4(S445N) cell that was photographed at 10-s intervals 10 min after plating. Panel iv shows an example of a PAK4(S445N) cell that had already begun to spread onto the fibronectin-coated surface 1 h after plating. Panels v and vi show control cells 10 min and 1 h, respectively, after plating. (C) Morphological changes in Rat1 cells overexpressing PAK(S445N). Top panels, cells containing either PAK4(S445N) or empty vector (pLPC) were visualized by phase-contrast microscopy under a 10× objective 24 h after plating; middle panels, cells were fixed 24 h after plating onto fibronectin-coated coverslips, and polymerized actin was visualized under a 60× oil lens after staining with FITC-conjugated phalloidin; bottom panels, cells were fixed 24 h after plating, and focal adhesions were visualized by immunofluorescence microscopy under a 60× oil lens after staining with anti-vinculin antibody and rhodamine-conjugated secondary antibody. (D) Morphological changes in NIH 3T3 cells expressing PAK4(S445N). NIH 3T3 cells were analyzed as described for panel C.

    Techniques Used: Stable Transfection, Expressing, Western Blot, Plasmid Preparation, Microscopy, Staining, Immunofluorescence

    20) Product Images from "?-synuclein induced synapse damage is enhanced by amyloid-?1-42"

    Article Title: ?-synuclein induced synapse damage is enhanced by amyloid-?1-42

    Journal: Molecular Neurodegeneration

    doi: 10.1186/1750-1326-5-55

    αSN triggered the loss of synaptophysin from cultured neurons . (A) The synaptophysin content of cortical neurons incubated for 24 hours with αSN (●) or βSN (○) as shown. Values shown are the mean amount of synaptophysin (units) ± SD, n = 15. (B) Immunoblots showing the amount of synaptophysin and β-actin in extracts from cortical neurons that had been incubated for 24 hours with αSN as shown. (C) The synaptophysin content of hippocampal neurons incubated for 24 hours with αSN (●) or βSN (○) as shown. Values shown are the mean amount of synaptophysin (units) ± SD, n = 12.
    Figure Legend Snippet: αSN triggered the loss of synaptophysin from cultured neurons . (A) The synaptophysin content of cortical neurons incubated for 24 hours with αSN (●) or βSN (○) as shown. Values shown are the mean amount of synaptophysin (units) ± SD, n = 15. (B) Immunoblots showing the amount of synaptophysin and β-actin in extracts from cortical neurons that had been incubated for 24 hours with αSN as shown. (C) The synaptophysin content of hippocampal neurons incubated for 24 hours with αSN (●) or βSN (○) as shown. Values shown are the mean amount of synaptophysin (units) ± SD, n = 12.

    Techniques Used: Cell Culture, Incubation, Western Blot

    21) Product Images from "Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress"

    Article Title: Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006132

    BAG3 interacts with eVP40 in a PPxY-dependent manner. A) Extracts from HEK293T cells transfected with eVP40 or eVP40-ΔPT/PY plus BAG3-WT were first immunoprecipitated (IP) with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using mouse anti- myc antiserum. Expression controls for eVP40-WT, eVP40-ΔPT/PY, His-myc-tagged BAG3 and GAPDH are shown. B) Extracts from HEK293T cells transfected with Flag-tagged mVP40 or mVP40(P > A) plus BAG3-WT were first immunoprecipitated (IP) with either normal mouse IgG or anti-Flag antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using rabbit anti-His antiserum. Expression controls for mVP40, mVP40(P > A), His-myc-tagged BAG3 and β-actin are shown. C) Extracts from HEK293T cells transfected with eVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for eVP40-WT, endogenous BAG3, and GAPDH are shown. D) Extracts from HEK293T cells transfected with mVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or anti-mVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for mVP40-WT, endogenous BAG3, and β-actin are shown.
    Figure Legend Snippet: BAG3 interacts with eVP40 in a PPxY-dependent manner. A) Extracts from HEK293T cells transfected with eVP40 or eVP40-ΔPT/PY plus BAG3-WT were first immunoprecipitated (IP) with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using mouse anti- myc antiserum. Expression controls for eVP40-WT, eVP40-ΔPT/PY, His-myc-tagged BAG3 and GAPDH are shown. B) Extracts from HEK293T cells transfected with Flag-tagged mVP40 or mVP40(P > A) plus BAG3-WT were first immunoprecipitated (IP) with either normal mouse IgG or anti-Flag antisera as indicated. BAG3 was detected in the precipitates by Western blot (WB) using rabbit anti-His antiserum. Expression controls for mVP40, mVP40(P > A), His-myc-tagged BAG3 and β-actin are shown. C) Extracts from HEK293T cells transfected with eVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or polyclonal anti-eVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for eVP40-WT, endogenous BAG3, and GAPDH are shown. D) Extracts from HEK293T cells transfected with mVP40-WT alone were first immunoprecipitated with either normal rabbit IgG or anti-mVP40 antisera as indicated. Endogenous BAG3 was detected in the precipitates by Western blot using polyclonal anti-BAG3 antiserum. Expression controls for mVP40-WT, endogenous BAG3, and β-actin are shown.

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Expressing

    BAG3 sequesters VP40 away from the plasma membrane. HEK293T cells were mock-transfected or transfected with eVP40 (A) or mVP40 (C) plus either BAG3-WT, or BAG3-ΔN as indicated. Cytosol and plasma membrane (PM) fractions were isolated at 24 hrs post-transfection, and the indicated proteins were detected by Western blotting. β-actin served as a control protein for the cytosol fraction, whereas Na/K ATPase served as a control protein for the PM fraction. The amount of VP40 in the PM fraction in control cells (lanes 6) was set at 100% (bar graph). Quantification of the relative amount of PM-associated eVP40 (B) or mVP40 (D) from three independent experiments is shown. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p
    Figure Legend Snippet: BAG3 sequesters VP40 away from the plasma membrane. HEK293T cells were mock-transfected or transfected with eVP40 (A) or mVP40 (C) plus either BAG3-WT, or BAG3-ΔN as indicated. Cytosol and plasma membrane (PM) fractions were isolated at 24 hrs post-transfection, and the indicated proteins were detected by Western blotting. β-actin served as a control protein for the cytosol fraction, whereas Na/K ATPase served as a control protein for the PM fraction. The amount of VP40 in the PM fraction in control cells (lanes 6) was set at 100% (bar graph). Quantification of the relative amount of PM-associated eVP40 (B) or mVP40 (D) from three independent experiments is shown. Statistical significance was analyzed by one-way ANOVA. ns: not significant, *** = p

    Techniques Used: Transfection, Isolation, Western Blot

    22) Product Images from "In Vivo Imaging of Brain Ischemia Using an Oxygen-Dependent Degradative Fusion Protein Probe"

    Article Title: In Vivo Imaging of Brain Ischemia Using an Oxygen-Dependent Degradative Fusion Protein Probe

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048051

    HIF-1α accumulation after focal brain ischemia. (A) Western blot analysis of HIF-1α in the ischemic and non-ischemic hemispheres of mice subjected to MCAO followed by reperfusion. (B) Densitometric analysis of HIF-1α protein levels in the ischemic hemispheres. Data were normalized relative to β-actin levels, and the values obtained from sham-operated controls (S) were arbitrarily defined as 1. * P
    Figure Legend Snippet: HIF-1α accumulation after focal brain ischemia. (A) Western blot analysis of HIF-1α in the ischemic and non-ischemic hemispheres of mice subjected to MCAO followed by reperfusion. (B) Densitometric analysis of HIF-1α protein levels in the ischemic hemispheres. Data were normalized relative to β-actin levels, and the values obtained from sham-operated controls (S) were arbitrarily defined as 1. * P

    Techniques Used: Western Blot, Mouse Assay

    23) Product Images from "RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1"

    Article Title: RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms160817611

    Effect of caspase-resistant Beclin-1 on autophagy and apoptosis in Beclin-1 knock down cells. ( A ) Endogenous Beclin-1 levels were knocked down using siRNA that does not target Beclin-1 cDNA and expression of RNAi-resistant Flag-Beclin-1 (WT) or RNAi and caspase-resistant Flag-Beclin-1 D133A/D149A was detected on immunoblots and normalized to β-actin levels; ( B ) Knock-in cells expressing GFP-LC3 were transfected with 2–5A (10 µM), for indicated times and the percentage of GFP + cells showing puncta formation compared to mock treated cells was analyzed. Student’s t test was used to determine p values of knock-in cells expressing WT Beclin-1 compared to caspase-resistant Beclin-1. * p
    Figure Legend Snippet: Effect of caspase-resistant Beclin-1 on autophagy and apoptosis in Beclin-1 knock down cells. ( A ) Endogenous Beclin-1 levels were knocked down using siRNA that does not target Beclin-1 cDNA and expression of RNAi-resistant Flag-Beclin-1 (WT) or RNAi and caspase-resistant Flag-Beclin-1 D133A/D149A was detected on immunoblots and normalized to β-actin levels; ( B ) Knock-in cells expressing GFP-LC3 were transfected with 2–5A (10 µM), for indicated times and the percentage of GFP + cells showing puncta formation compared to mock treated cells was analyzed. Student’s t test was used to determine p values of knock-in cells expressing WT Beclin-1 compared to caspase-resistant Beclin-1. * p

    Techniques Used: Expressing, Western Blot, Knock-In, Transfection

    Regulation of autophagy by inhibiting dsRNA-induced apoptosis. HT1080 cells were pretreated with zVAD-FMK (20 µM) or not for 1h followed by 2 µg/mL of PolyI:C for indicated times. ( A ) Cleavage of caspase 3 was determined on immunoblots and normalized to β-actin levels; ( B ) cell viability was determined by MTT assay and normalized to control cells. Results are representative of three independent experiments performed in triplicate ± SD; ( C ) Conversion of unconjugated LC3-I to lipidated LC3-II and degradation of p62 was monitored on immunoblots and normalized to β-actin levels. Band intensity was calculated using Image J software and ratios of LC3-II/β-actin was determined. Results are representative of three independent experiments; ( D ) GFP-LC3 expressing HT1080 cells were pretreated with zVAD-FMK (20 µM) or not for 1 h followed by 2 µg/mL of PolyI:C or mock treated for indicated times. The percentage of GFP + cells showing puncta formation compared to mock treated cells was analyzed. Results shown represent mean ± SEM for three experiments and at least 100 cells were analyzed per assay.
    Figure Legend Snippet: Regulation of autophagy by inhibiting dsRNA-induced apoptosis. HT1080 cells were pretreated with zVAD-FMK (20 µM) or not for 1h followed by 2 µg/mL of PolyI:C for indicated times. ( A ) Cleavage of caspase 3 was determined on immunoblots and normalized to β-actin levels; ( B ) cell viability was determined by MTT assay and normalized to control cells. Results are representative of three independent experiments performed in triplicate ± SD; ( C ) Conversion of unconjugated LC3-I to lipidated LC3-II and degradation of p62 was monitored on immunoblots and normalized to β-actin levels. Band intensity was calculated using Image J software and ratios of LC3-II/β-actin was determined. Results are representative of three independent experiments; ( D ) GFP-LC3 expressing HT1080 cells were pretreated with zVAD-FMK (20 µM) or not for 1 h followed by 2 µg/mL of PolyI:C or mock treated for indicated times. The percentage of GFP + cells showing puncta formation compared to mock treated cells was analyzed. Results shown represent mean ± SEM for three experiments and at least 100 cells were analyzed per assay.

    Techniques Used: Western Blot, MTT Assay, Software, Expressing

    Cleavage of Beclin-1 in RNase L-mediated cross-talk between autophagy and apoptosis. HT1080 cells were transfected with 10 µM of 2–5A or 2 µg/mL of PolyI:C and ( A ) RNase L-mediated cleavage of rRNA (arrows) was analyzed on RNA chips using the Agilent Bioanalyzer 2100 after 6 h. Cell viability was determined at indicated times by ( B ) MTT colorimetric assays, ( C ) trypan blue dye exclusion assay normalized to control cells or ( D ) uptake of PI by dying cells as measured by flow cytometry after staining with PI. Results are representative of three independent experiments performed in triplicate ± SD; ( E ) Cleavage of Caspase 3 and PARP in cell lysates from 2–5A or PolyI:C transfected cells was analyzed on immunoblots and normalized to β-actin levels; ( F ) Caspase 3/7 activity was measured in 2–5A transfected cells at indicated times using rhodamine-labeled caspase-3 and -7 substrate (ApoONE homogenous caspase-3 and -7 assay kit (Promega). Results are representative of three independent experiments performed in triplicate ± SD; ( G ) GFP-LC3 expressing HT1080 cells were mock treated, transfected with 10 µM of 2–5A or 2 µg/mL of PolyI:C for indicated times and the percentage of GFP + cells showing puncta formation compared to mock treated cells was analyzed. Results shown represent mean ± SEM for three experiments and at least 100 cells were analyzed per assay, p values are shown as compared with mock treated cells; ( H ) Cleavage of Beclin-1 was monitored in response to 2–5A or PolyI:C for indicated times on immunoblots and normalized to β-actin levels; ( I ) HT1080 cells expressing Flag-Beclin-1 were pretreated with zVAD-FMK (20 µM) or not for 1 h followed by 2 µg/mL of PolyI:C for indicated times. Cleavage of Beclin-1 was determined on immunoblots and normalized to β-actin levels. Results are representative of three independent experiments. Student’s t test was used to determine p values. * p
    Figure Legend Snippet: Cleavage of Beclin-1 in RNase L-mediated cross-talk between autophagy and apoptosis. HT1080 cells were transfected with 10 µM of 2–5A or 2 µg/mL of PolyI:C and ( A ) RNase L-mediated cleavage of rRNA (arrows) was analyzed on RNA chips using the Agilent Bioanalyzer 2100 after 6 h. Cell viability was determined at indicated times by ( B ) MTT colorimetric assays, ( C ) trypan blue dye exclusion assay normalized to control cells or ( D ) uptake of PI by dying cells as measured by flow cytometry after staining with PI. Results are representative of three independent experiments performed in triplicate ± SD; ( E ) Cleavage of Caspase 3 and PARP in cell lysates from 2–5A or PolyI:C transfected cells was analyzed on immunoblots and normalized to β-actin levels; ( F ) Caspase 3/7 activity was measured in 2–5A transfected cells at indicated times using rhodamine-labeled caspase-3 and -7 substrate (ApoONE homogenous caspase-3 and -7 assay kit (Promega). Results are representative of three independent experiments performed in triplicate ± SD; ( G ) GFP-LC3 expressing HT1080 cells were mock treated, transfected with 10 µM of 2–5A or 2 µg/mL of PolyI:C for indicated times and the percentage of GFP + cells showing puncta formation compared to mock treated cells was analyzed. Results shown represent mean ± SEM for three experiments and at least 100 cells were analyzed per assay, p values are shown as compared with mock treated cells; ( H ) Cleavage of Beclin-1 was monitored in response to 2–5A or PolyI:C for indicated times on immunoblots and normalized to β-actin levels; ( I ) HT1080 cells expressing Flag-Beclin-1 were pretreated with zVAD-FMK (20 µM) or not for 1 h followed by 2 µg/mL of PolyI:C for indicated times. Cleavage of Beclin-1 was determined on immunoblots and normalized to β-actin levels. Results are representative of three independent experiments. Student’s t test was used to determine p values. * p

    Techniques Used: Transfection, MTT Assay, Exclusion Assay, Flow Cytometry, Cytometry, Staining, Western Blot, Activity Assay, Labeling, Expressing

    Cells lacking autophagy proteins undergo apoptosis in response to RNase L activation. ( A ) HT1080 cells were transfected with Beclin siRNAs (20 nM) or control siRNAs (20 nM) and knock down of Beclin-1 protein levels were determined on immunoblots; ( B ) Control or Beclin-1 siRNA expressing cells were transfected with 2–5A (10 µM) and cell viability was determined using MTT assay at indicated times. Results are representative of three independent experiments performed in triplicate ± SD; ( C ) Cleavage of PARP (indicated by arrow) and caspase 3 was monitored in cell lysates of 2–5A treated knock-down cells and compared to control cells. Protein levels were and normalized to β-actin; ( D ) WT or Atg5 KO MEFs were transfected with 10 µM of 2–5A for indicated times and induction of apoptosis was monitored by cleavage of PARP (indicated by arrow) and cleaved caspase 3 on immunoblots normalized to β-actin levels. Results are representative of three independent experiments.
    Figure Legend Snippet: Cells lacking autophagy proteins undergo apoptosis in response to RNase L activation. ( A ) HT1080 cells were transfected with Beclin siRNAs (20 nM) or control siRNAs (20 nM) and knock down of Beclin-1 protein levels were determined on immunoblots; ( B ) Control or Beclin-1 siRNA expressing cells were transfected with 2–5A (10 µM) and cell viability was determined using MTT assay at indicated times. Results are representative of three independent experiments performed in triplicate ± SD; ( C ) Cleavage of PARP (indicated by arrow) and caspase 3 was monitored in cell lysates of 2–5A treated knock-down cells and compared to control cells. Protein levels were and normalized to β-actin; ( D ) WT or Atg5 KO MEFs were transfected with 10 µM of 2–5A for indicated times and induction of apoptosis was monitored by cleavage of PARP (indicated by arrow) and cleaved caspase 3 on immunoblots normalized to β-actin levels. Results are representative of three independent experiments.

    Techniques Used: Activation Assay, Transfection, Western Blot, Expressing, MTT Assay

    24) Product Images from "The Herpes Simplex Virus 2 Virion-Associated Ribonuclease vhs Interferes with Stress Granule Formation"

    Article Title: The Herpes Simplex Virus 2 Virion-Associated Ribonuclease vhs Interferes with Stress Granule Formation

    Journal: Journal of Virology

    doi: 10.1128/JVI.01554-14

    Spontaneous SGs contain viral proteins. HeLa cells were infected at high MOI with FS virus. Seven hours later, cells were fixed and stained with goat polyclonal antiserum specific for TIA-1 and mouse monoclonal antibody specific for ICP27, ICP8, or gC or with rat polyclonal antiserum specific for Us3 followed by staining with Alexa Fluor 488- or Alexa Fluor 568-conjugated donkey anti-goat IgG and Alexa Fluor 568-conjugated donkey anti-mouse IgG secondary antibodies or Alexa Fluor 488-conjugated donkey anti-rat IgG antibodies. Nuclei were stained with Hoechst 33342. Stained cells were examined by confocal microscopy, and representative images are shown. The percentage of colocalization observed between TIA-1 and viral protein in approximately 200 spontaneous SGs is shown on the merged image panels. Scale bars, 10 μm. The graphs on the right show the fluorescence intensity across an approximately 8-μm line indicated on the merged image panels. For comparative purposes, the confocal acquisition settings used for the secondary-antibody-alone samples were the same as those used for the corresponding viral protein(s).
    Figure Legend Snippet: Spontaneous SGs contain viral proteins. HeLa cells were infected at high MOI with FS virus. Seven hours later, cells were fixed and stained with goat polyclonal antiserum specific for TIA-1 and mouse monoclonal antibody specific for ICP27, ICP8, or gC or with rat polyclonal antiserum specific for Us3 followed by staining with Alexa Fluor 488- or Alexa Fluor 568-conjugated donkey anti-goat IgG and Alexa Fluor 568-conjugated donkey anti-mouse IgG secondary antibodies or Alexa Fluor 488-conjugated donkey anti-rat IgG antibodies. Nuclei were stained with Hoechst 33342. Stained cells were examined by confocal microscopy, and representative images are shown. The percentage of colocalization observed between TIA-1 and viral protein in approximately 200 spontaneous SGs is shown on the merged image panels. Scale bars, 10 μm. The graphs on the right show the fluorescence intensity across an approximately 8-μm line indicated on the merged image panels. For comparative purposes, the confocal acquisition settings used for the secondary-antibody-alone samples were the same as those used for the corresponding viral protein(s).

    Techniques Used: Infection, Staining, Confocal Microscopy, Fluorescence

    25) Product Images from "Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins"

    Article Title: Phosphorylated Human Keratinocyte Ornithine Decarboxylase Is Preferentially Associated with Insoluble Cellular Proteins

    Journal: Molecular Biology of the Cell

    doi:

    ODC and cytoskeletal organization in cytochalasin D–treated NHEK. To determine the effects of remodeling the cytoskeleton on ODC organization, NHEK were treated with 1 μg/ml cytochalasin D for 6 h, stained for ODC (B, E, or H) and actin (A), tubulin (D), or keratin (G), and optically sectioned by confocal laser-scanning microscopy, and corresponding images were superimposed to determine the degrees of overlap (C, F, or I; orange-yellow). Superposition of actin (A) with ODC (B) showed only minimal areas of overlap (C). Tubulin (D) and ODC (E) staining showed nonspecific areas of overlap (F). Keratin (G) and ODC (H) exhibited extensive overlap throughout the cells (I). Bars: A–C, D–F, and G–I, 10 μm.
    Figure Legend Snippet: ODC and cytoskeletal organization in cytochalasin D–treated NHEK. To determine the effects of remodeling the cytoskeleton on ODC organization, NHEK were treated with 1 μg/ml cytochalasin D for 6 h, stained for ODC (B, E, or H) and actin (A), tubulin (D), or keratin (G), and optically sectioned by confocal laser-scanning microscopy, and corresponding images were superimposed to determine the degrees of overlap (C, F, or I; orange-yellow). Superposition of actin (A) with ODC (B) showed only minimal areas of overlap (C). Tubulin (D) and ODC (E) staining showed nonspecific areas of overlap (F). Keratin (G) and ODC (H) exhibited extensive overlap throughout the cells (I). Bars: A–C, D–F, and G–I, 10 μm.

    Techniques Used: Staining, Confocal Laser Scanning Microscopy

    ODC and cytoskeletal organization in normal human epidermal keratinocytes. To assess potential colocalization of ODC with a cytoskeletal component, NHEK were stained for ODC (B, E, or H) and actin (A), tubulin (D), or keratin (G) and optically sectioned using confocal laser-scanning microscopy, and corresponding images were superimposed to determine the degrees of overlap (C, F, or I; orange-yellow→yellow). Superposition of actin (A) with ODC (B) showed little overlap (C) of the two staining patterns. Tubulin (D) and ODC (E) staining showed little overlap (F) except in a cell undergoing mitosis in which staining was apparent throughout. Keratin (G) and ODC (H) exhibited overlap (I) in the perinuclear region of the cell with little overlap outside of that region. Digital micrographs were collected using the Noran Intervision software (Noran Instruments). Bars: A–C, D–F, and G–I, 10 μm.
    Figure Legend Snippet: ODC and cytoskeletal organization in normal human epidermal keratinocytes. To assess potential colocalization of ODC with a cytoskeletal component, NHEK were stained for ODC (B, E, or H) and actin (A), tubulin (D), or keratin (G) and optically sectioned using confocal laser-scanning microscopy, and corresponding images were superimposed to determine the degrees of overlap (C, F, or I; orange-yellow→yellow). Superposition of actin (A) with ODC (B) showed little overlap (C) of the two staining patterns. Tubulin (D) and ODC (E) staining showed little overlap (F) except in a cell undergoing mitosis in which staining was apparent throughout. Keratin (G) and ODC (H) exhibited overlap (I) in the perinuclear region of the cell with little overlap outside of that region. Digital micrographs were collected using the Noran Intervision software (Noran Instruments). Bars: A–C, D–F, and G–I, 10 μm.

    Techniques Used: Staining, Confocal Laser Scanning Microscopy, Software

    26) Product Images from "Functional and Structural Analyses of CYP1B1 Variants Linked to Congenital and Adult-Onset Glaucoma to Investigate the Molecular Basis of These Diseases"

    Article Title: Functional and Structural Analyses of CYP1B1 Variants Linked to Congenital and Adult-Onset Glaucoma to Investigate the Molecular Basis of These Diseases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0156252

    Protein turnover rate of CYP1B1 constructs. Transfected HEK 293T cells were treated with CHX for 12h to inhibit protein synthesis. Twenty μg cell extracts were probed sequentially, by western blot analysis, with appropriate antibodies: Myc (to detect recombinant CYP1B1) Cell Signaling Technology, USA] and β-actin (to serve as a loading control) (Sigma, USA). Immunoblots were scanned, and net pixel intensities of the bands were measured with Image J software. CYP1B1 values were normalized to β-actin, the mean values were taken for three separate transfections, and the relative amounts of CYP1B1 were expressed as a percentage of levels of WT at respective time points. Representative lanes from western blots are furnished on the right side of the panels. Panel A : Mutations found in only POAG cases; Panel B : Mutations found in both POAG and PCG cases; Panel C : Mutations found in only PCG cases. Level of WT protein under similar conditions is shown in panel A. Data represent the mean ± SEM for a triplicate per group. Data were tested by an unpaired t test. Differences in mean were assessed for statistical significance ( p
    Figure Legend Snippet: Protein turnover rate of CYP1B1 constructs. Transfected HEK 293T cells were treated with CHX for 12h to inhibit protein synthesis. Twenty μg cell extracts were probed sequentially, by western blot analysis, with appropriate antibodies: Myc (to detect recombinant CYP1B1) Cell Signaling Technology, USA] and β-actin (to serve as a loading control) (Sigma, USA). Immunoblots were scanned, and net pixel intensities of the bands were measured with Image J software. CYP1B1 values were normalized to β-actin, the mean values were taken for three separate transfections, and the relative amounts of CYP1B1 were expressed as a percentage of levels of WT at respective time points. Representative lanes from western blots are furnished on the right side of the panels. Panel A : Mutations found in only POAG cases; Panel B : Mutations found in both POAG and PCG cases; Panel C : Mutations found in only PCG cases. Level of WT protein under similar conditions is shown in panel A. Data represent the mean ± SEM for a triplicate per group. Data were tested by an unpaired t test. Differences in mean were assessed for statistical significance ( p

    Techniques Used: Construct, Transfection, Western Blot, Recombinant, Software

    27) Product Images from "Itm2a silencing rescues lamin A mediated inhibition of 3T3-L1 adipocyte differentiation"

    Article Title: Itm2a silencing rescues lamin A mediated inhibition of 3T3-L1 adipocyte differentiation

    Journal: Adipocyte

    doi: 10.1080/21623945.2017.1362510

    shRNA mediated knockdown of Itm2a enhances 3T3-L1 differentiation and increases PPARγ protein. 3T3-L1 preadipocytes were stably transfected with pRFP(PB).shItm2a or scramble control pRFP(PB).shControl and induced to differentiate using full induction media MDI (methylisobutylxanthine, dexamethasone and insulin), sub-maximal media DI (dexamethasone and insulin) or D (dexamethasone) as indicated. (A) Adipogenesis was assessed at day 8 post induction by staining with Oil Red O; quantification was performed using ImageJ and expressed as Oil Red O absorbance units (ORO a.u.). (B, D) qPCR analysis of Itm2a, PPARγ and CEBPα and immunoblot analysis of PPARγ (E,F) expression during differentiation of 3T3-L1 cells stably transfected with pRFP(PB).shItm2a or scramble control pRFP(PB). (C) Immunoblot analysis of ITM2A knockdown in 3T3-NIH cells dual transfected with pCMV.Itm2a and pRFP(PB).shItm2a or pRFP(PB).sh.Control. (G) Quantification of PPARγ protein isoforms relative to β-actin, with mean and standard deviations determined by densitometry from 2 biologic replicates. Statistical significance compared with scramble control cells indicated as follows: * = P
    Figure Legend Snippet: shRNA mediated knockdown of Itm2a enhances 3T3-L1 differentiation and increases PPARγ protein. 3T3-L1 preadipocytes were stably transfected with pRFP(PB).shItm2a or scramble control pRFP(PB).shControl and induced to differentiate using full induction media MDI (methylisobutylxanthine, dexamethasone and insulin), sub-maximal media DI (dexamethasone and insulin) or D (dexamethasone) as indicated. (A) Adipogenesis was assessed at day 8 post induction by staining with Oil Red O; quantification was performed using ImageJ and expressed as Oil Red O absorbance units (ORO a.u.). (B, D) qPCR analysis of Itm2a, PPARγ and CEBPα and immunoblot analysis of PPARγ (E,F) expression during differentiation of 3T3-L1 cells stably transfected with pRFP(PB).shItm2a or scramble control pRFP(PB). (C) Immunoblot analysis of ITM2A knockdown in 3T3-NIH cells dual transfected with pCMV.Itm2a and pRFP(PB).shItm2a or pRFP(PB).sh.Control. (G) Quantification of PPARγ protein isoforms relative to β-actin, with mean and standard deviations determined by densitometry from 2 biologic replicates. Statistical significance compared with scramble control cells indicated as follows: * = P

    Techniques Used: shRNA, Stable Transfection, Transfection, Staining, Real-time Polymerase Chain Reaction, Expressing

    Itm2a overexpression in 3T3-L1 differentiation. 3T3-L1 preadipocytes were stably transfected with pMSCV(PB)-Itm2a or empty vector control pMSCV(PB) plasmid and induced to differentiate into adipocytes. (A) Adipogenesis was assessed at day 8 post induction by staining with Oil Red O and quantification was performed using ImageJ and expressed as Oil red O absorbance units (ORO a.u.). (B,C) qPCR analysis of Itm2a and PPARγ expression during differentiation of stably transfected 3T3-L1 cells. (D) Immunoblot analysis of PPARγ at day 4 post induction. (E) Quantification of PPARγ protein relative to β-actin, with mean and standard deviations determined by densitometry from 2 biologic replicates. A Student's t -test (2-tailed, assuming equal variance) was used to calculate statistical significance compared with empty vector control cells, indicated as follows: * = P
    Figure Legend Snippet: Itm2a overexpression in 3T3-L1 differentiation. 3T3-L1 preadipocytes were stably transfected with pMSCV(PB)-Itm2a or empty vector control pMSCV(PB) plasmid and induced to differentiate into adipocytes. (A) Adipogenesis was assessed at day 8 post induction by staining with Oil Red O and quantification was performed using ImageJ and expressed as Oil red O absorbance units (ORO a.u.). (B,C) qPCR analysis of Itm2a and PPARγ expression during differentiation of stably transfected 3T3-L1 cells. (D) Immunoblot analysis of PPARγ at day 4 post induction. (E) Quantification of PPARγ protein relative to β-actin, with mean and standard deviations determined by densitometry from 2 biologic replicates. A Student's t -test (2-tailed, assuming equal variance) was used to calculate statistical significance compared with empty vector control cells, indicated as follows: * = P

    Techniques Used: Over Expression, Stable Transfection, Transfection, Plasmid Preparation, Staining, Real-time Polymerase Chain Reaction, Expressing

    28) Product Images from "EFFECTS OF AGE AND INSULIN-LIKE GROWTH FACTOR-1 ON RAT NEUROTROPHIN RECEPTOR EXPRESSION AFTER NERVE INJURY"

    Article Title: EFFECTS OF AGE AND INSULIN-LIKE GROWTH FACTOR-1 ON RAT NEUROTROPHIN RECEPTOR EXPRESSION AFTER NERVE INJURY

    Journal: Muscle & nerve

    doi: 10.1002/mus.25106

    Western blot results of IGF-1R, p75 NTR , sortilin, and TrkB with β -actin as loading control. Results are shown for uninjured, injured at 1, 3, and 10 days in aged and young rats.
    Figure Legend Snippet: Western blot results of IGF-1R, p75 NTR , sortilin, and TrkB with β -actin as loading control. Results are shown for uninjured, injured at 1, 3, and 10 days in aged and young rats.

    Techniques Used: Western Blot

    29) Product Images from "Recombinant Resilin-based Bioelastomers for Regenerative Medicine Applications"

    Article Title: Recombinant Resilin-based Bioelastomers for Regenerative Medicine Applications

    Journal: Advanced healthcare materials

    doi: 10.1002/adhm.201500411

    Immunochemical analysis of hMSCs stained, after 72 hours, for nuclei, vinculin, and actin cytoskeleton, on the surface of 20wt% RLP hydrogels
    Figure Legend Snippet: Immunochemical analysis of hMSCs stained, after 72 hours, for nuclei, vinculin, and actin cytoskeleton, on the surface of 20wt% RLP hydrogels

    Techniques Used: Staining

    30) Product Images from "Analysis artefacts of the INS-IGF2 fusion transcript"

    Article Title: Analysis artefacts of the INS-IGF2 fusion transcript

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-015-0042-8

    Protein level sequence comparison between unprocessed preproinsulin and the INS-IGF2 fusion protein. The epitope of the proinsulin specific monoclonal antibody GS-9A8 is indicated [ 4 ]. The BO1P antibody also used in this study was raised against the full length INS-IGF2 protein.
    Figure Legend Snippet: Protein level sequence comparison between unprocessed preproinsulin and the INS-IGF2 fusion protein. The epitope of the proinsulin specific monoclonal antibody GS-9A8 is indicated [ 4 ]. The BO1P antibody also used in this study was raised against the full length INS-IGF2 protein.

    Techniques Used: Sequencing

    Immunoblot analysis and immunostaining of human pancreas. a Western blots investigating the presence of proinsulin-immunoreactivity (using antibody GS-9A8, upper panel ) and INS-IGF2 fusion protein immunoreactivity (using antibody BO1P, lower panel ) in the human beta cell line EndoC-βH1 and HEK, non transduced, GFP transduced or INS-IGF2 transduced. Endogenous proinsulin is marked with ( Asterisk ) and INS-IGF2 with ( filled circle ). Notice that INS-IGF2 is only reliably detected in cells transduced with INS-IGF2 construct. We titrated the levels of transduced HEK293 cell extract to give comparable band intensities on EndoC-βH1 proinsulin and HEK293-INS-IGF2 ( panel a upper : GS-9A8 which has assumed identical affinities to the two proteins). INS-IGF2 transduction of EndoC-βH1 leads to relative lower expression of INS-IGF2-protein compared to proinsulin ( panel a upper comparing the two bands in lane INS-IGF2-EndoC-βH1). This low-level expression of INS-IGF2 protein in transduced EndoC-βH1 is readily detected using the INS-IGF2 antibody while untransduced cells are completely negative ( panel a lower ). Thus we conclude that the expression of INS-IGF2 protein in EndoC-βH1 is below detection limits of this assay. b Immunoreactivity for INS-IGF2 ( green ) and glucagon ( red ) ( top panel ), and for proinsulin ( green ) and glucagon ( red ) ( bottom panel ) on adjacent sections of human pancreas. Scale bar 100 µM.
    Figure Legend Snippet: Immunoblot analysis and immunostaining of human pancreas. a Western blots investigating the presence of proinsulin-immunoreactivity (using antibody GS-9A8, upper panel ) and INS-IGF2 fusion protein immunoreactivity (using antibody BO1P, lower panel ) in the human beta cell line EndoC-βH1 and HEK, non transduced, GFP transduced or INS-IGF2 transduced. Endogenous proinsulin is marked with ( Asterisk ) and INS-IGF2 with ( filled circle ). Notice that INS-IGF2 is only reliably detected in cells transduced with INS-IGF2 construct. We titrated the levels of transduced HEK293 cell extract to give comparable band intensities on EndoC-βH1 proinsulin and HEK293-INS-IGF2 ( panel a upper : GS-9A8 which has assumed identical affinities to the two proteins). INS-IGF2 transduction of EndoC-βH1 leads to relative lower expression of INS-IGF2-protein compared to proinsulin ( panel a upper comparing the two bands in lane INS-IGF2-EndoC-βH1). This low-level expression of INS-IGF2 protein in transduced EndoC-βH1 is readily detected using the INS-IGF2 antibody while untransduced cells are completely negative ( panel a lower ). Thus we conclude that the expression of INS-IGF2 protein in EndoC-βH1 is below detection limits of this assay. b Immunoreactivity for INS-IGF2 ( green ) and glucagon ( red ) ( top panel ), and for proinsulin ( green ) and glucagon ( red ) ( bottom panel ) on adjacent sections of human pancreas. Scale bar 100 µM.

    Techniques Used: Immunostaining, Western Blot, Transduction, Construct, Expressing

    31) Product Images from "Biocompatibility of Polypyrrole with Human Primary Osteoblasts and the Effect of Dopants"

    Article Title: Biocompatibility of Polypyrrole with Human Primary Osteoblasts and the Effect of Dopants

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134023

    Focal adhesion and attachment. Vinculin attachment points / 100 μm 2 cell area [A] and percentage share of vinculin area / cell area [B] was analyzed with Fiji ImageJ. The value represents mean ± standard deviation (n = 27). *p
    Figure Legend Snippet: Focal adhesion and attachment. Vinculin attachment points / 100 μm 2 cell area [A] and percentage share of vinculin area / cell area [B] was analyzed with Fiji ImageJ. The value represents mean ± standard deviation (n = 27). *p

    Techniques Used: Standard Deviation

    Vinculin staining. Representative vinculin staining of attachment points, visualized with Alexa Flour 488(green) and F-actin filament staining with Alexa Fluor 568 phalloidin(red) of gold [A] and Polypyrrole (PPy) doped with chondroitin sulfate (CS) [B], dodecylbenzenesulfonate (DBS) [C] and p-Toluene sulfonate (pTS) [D]. The magnification is 63-fold.
    Figure Legend Snippet: Vinculin staining. Representative vinculin staining of attachment points, visualized with Alexa Flour 488(green) and F-actin filament staining with Alexa Fluor 568 phalloidin(red) of gold [A] and Polypyrrole (PPy) doped with chondroitin sulfate (CS) [B], dodecylbenzenesulfonate (DBS) [C] and p-Toluene sulfonate (pTS) [D]. The magnification is 63-fold.

    Techniques Used: Staining

    32) Product Images from "Sorting of β-Actin mRNA and Protein to Neurites and Growth Cones in Culture"

    Article Title: Sorting of β-Actin mRNA and Protein to Neurites and Growth Cones in Culture

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.18-01-00251.1998

    ). A , Localization of β-actin mRNA within a single optical section (250 nm) of a cell body and minor processes (unprocessed image). The fluorescence intensity within the neurite shaft was low. A concentration of β-actin mRNA was observed within the growth cone. B , The same image after restoration. A punctate distribution was observed throughout most of the processes. Note the concentration of β-actin mRNA within one of the growth cones ( arrow ). C , D ) Localization of β-actin mRNA within an axon and its growth cone. The cell body is at the top of the image, and the axon extends downward, terminating in an elaborate growth cone. Note the concentration of β-actin mRNA granules within the central domain ( arrow ) and few granules within peripheral regions ( curved arrow ). Scale bar, 10 μm.
    Figure Legend Snippet: ). A , Localization of β-actin mRNA within a single optical section (250 nm) of a cell body and minor processes (unprocessed image). The fluorescence intensity within the neurite shaft was low. A concentration of β-actin mRNA was observed within the growth cone. B , The same image after restoration. A punctate distribution was observed throughout most of the processes. Note the concentration of β-actin mRNA within one of the growth cones ( arrow ). C , D ) Localization of β-actin mRNA within an axon and its growth cone. The cell body is at the top of the image, and the axon extends downward, terminating in an elaborate growth cone. Note the concentration of β-actin mRNA granules within the central domain ( arrow ) and few granules within peripheral regions ( curved arrow ). Scale bar, 10 μm.

    Techniques Used: Fluorescence, Concentration Assay

    Transport of β-actin mRNA into processes after treatment with db-cAMP. Cortical neurons cultured for 4 d in N2 supplements were transferred to MEM for 3 hr. Cells were fixed and hybridized with digoxigenin-labeled probes specific to β-actin mRNA. Probes were detected by using fluorochrome-conjugated antibodies, and images were acquired with a cooled CCD camera (see Materials and Methods). A , β-Actin mRNA was detected in the cell body, but the signal no longer was observed in growth cones ( arrow ). C , After 15 min in db-cAMP, β-actin mRNA granules were observed in processes ( arrow ) but were not yet detectable within growth cones ( arrowhead ). Shown here is a signal within an axonal process. E , After 1 hr, β-actin mRNA granules were observed within growth cones. Shown here is a hybridization signal within the distal axon ( arrowhead ) and growth cone ( arrow ). G , γ-Actin mRNA was confined to the cell body ( arrow ). No signal was observed within the axonal growth cone ( arrowhead ). Shown here is a cell after 1 hr of treatment with db-cAMP. B, D, F, H , DIC optics. Scale bar, 10 μm.
    Figure Legend Snippet: Transport of β-actin mRNA into processes after treatment with db-cAMP. Cortical neurons cultured for 4 d in N2 supplements were transferred to MEM for 3 hr. Cells were fixed and hybridized with digoxigenin-labeled probes specific to β-actin mRNA. Probes were detected by using fluorochrome-conjugated antibodies, and images were acquired with a cooled CCD camera (see Materials and Methods). A , β-Actin mRNA was detected in the cell body, but the signal no longer was observed in growth cones ( arrow ). C , After 15 min in db-cAMP, β-actin mRNA granules were observed in processes ( arrow ) but were not yet detectable within growth cones ( arrowhead ). Shown here is a signal within an axonal process. E , After 1 hr, β-actin mRNA granules were observed within growth cones. Shown here is a hybridization signal within the distal axon ( arrowhead ) and growth cone ( arrow ). G , γ-Actin mRNA was confined to the cell body ( arrow ). No signal was observed within the axonal growth cone ( arrowhead ). Shown here is a cell after 1 hr of treatment with db-cAMP. B, D, F, H , DIC optics. Scale bar, 10 μm.

    Techniques Used: Cell Culture, Labeling, Hybridization

    Localization of β-actin protein visualized in optical sections via image processing. Cortical neurons cultured for 4 d were double-labeled with phalloidin (rhodamine) and an isoform-specific antibody to β-actin (fluorescein). Images were superimposed after restoration and then registered (see Materials and Methods). Shown here is a single optical section (250 nm) from the z -series. A , Phalloidin labeled actin filaments throughout the cell body and neurites, whereas the β-actin isoform was concentrated within the distal tips of minor processes. The overlap is indicated by the presence of white pixels. B , In distal axons, phalloidin labeling is distributed throughout the neurite and growth cone, whereas the β-actin isoform is localized to the peripheral margin; note the apparent fibrillar distribution within filopodia. Scale bar, 10 μm.
    Figure Legend Snippet: Localization of β-actin protein visualized in optical sections via image processing. Cortical neurons cultured for 4 d were double-labeled with phalloidin (rhodamine) and an isoform-specific antibody to β-actin (fluorescein). Images were superimposed after restoration and then registered (see Materials and Methods). Shown here is a single optical section (250 nm) from the z -series. A , Phalloidin labeled actin filaments throughout the cell body and neurites, whereas the β-actin isoform was concentrated within the distal tips of minor processes. The overlap is indicated by the presence of white pixels. B , In distal axons, phalloidin labeling is distributed throughout the neurite and growth cone, whereas the β-actin isoform is localized to the peripheral margin; note the apparent fibrillar distribution within filopodia. Scale bar, 10 μm.

    Techniques Used: Cell Culture, Labeling

    Distance between β-actin mRNA granules and microtubules, as compared with randomized signals. The distance of β-actin mRNA (brightest voxels) to the nearest tubulin voxel was compared with a randomized distribution. This analysis was performed on a three-dimensional data set from 100 nm optical sections. The mean and SD of the random distribution are shown. The observed distribution of β-actin mRNA is significantly closer to the microtubules than a random distribution.
    Figure Legend Snippet: Distance between β-actin mRNA granules and microtubules, as compared with randomized signals. The distance of β-actin mRNA (brightest voxels) to the nearest tubulin voxel was compared with a randomized distribution. This analysis was performed on a three-dimensional data set from 100 nm optical sections. The mean and SD of the random distribution are shown. The observed distribution of β-actin mRNA is significantly closer to the microtubules than a random distribution.

    Techniques Used:

    Colocalization of β-actin mRNA and translational components. Shown are double labeling for β-actin mRNA (rhodamine) and EF1α or 60S ribosomes (fluorescein). A , EF1α and ( B ) β-actin mRNA in the distal field of an axonal process and its terminal branches. Punctate fluorescence for EF1α colocalized with β-actin mRNA granules ( arrows ). Image processing indicated that β-actin mRNA and EF1α occupied the same pixel coordinates. Note the identical spacing between the punctate distribution patterns in both rhodamine (β-actin mRNA) and fluorescein (EF1α). A granule containing EF1α does not colocalize with β-actin mRNA ( arrowheads ). C , D , Ribosomal subunit (60S) and actin mRNA in an axonal growth cone. C , Cluster of four granules that contain 60S protein ( arrow ) and ( D ) actin mRNA ( arrow ). A granule containing the 60S subunit does not contain actin mRNA ( arrowheads ). Scale bar, 5 μm.
    Figure Legend Snippet: Colocalization of β-actin mRNA and translational components. Shown are double labeling for β-actin mRNA (rhodamine) and EF1α or 60S ribosomes (fluorescein). A , EF1α and ( B ) β-actin mRNA in the distal field of an axonal process and its terminal branches. Punctate fluorescence for EF1α colocalized with β-actin mRNA granules ( arrows ). Image processing indicated that β-actin mRNA and EF1α occupied the same pixel coordinates. Note the identical spacing between the punctate distribution patterns in both rhodamine (β-actin mRNA) and fluorescein (EF1α). A granule containing EF1α does not colocalize with β-actin mRNA ( arrowheads ). C , D , Ribosomal subunit (60S) and actin mRNA in an axonal growth cone. C , Cluster of four granules that contain 60S protein ( arrow ) and ( D ) actin mRNA ( arrow ). A granule containing the 60S subunit does not contain actin mRNA ( arrowheads ). Scale bar, 5 μm.

    Techniques Used: Labeling, Fluorescence

    Localization of actin isoforms within cultured neurons. Cortical neurons cultured for 4 d were double-labeled with an isoform-specific antibody to β-actin or γ-actin ( left column ) and phalloidin–TRITC ( right column ). A , B , γ-Actin was distributed throughout the cell body ( arrow ) and neurites and resembled phalloidin staining. C , D , Localization of β-actin at tips of minor neurites ( arrowhead ). Low levels of β-actin were observed in the cell body ( arrow ). Phalloidin labeled actin filaments throughout the cell body ( arrow ) and minor neurites ( arrowhead ). E , F , γ-Actin was distributed throughout the axon and growth cone, as was phalloidin staining. G , H , β-Actin was enriched within axonal growth cones ( arrows ). Only weak labeling was observed in the axon shaft. Not all filopodia were labeled ( arrowheads ) despite the presence of F-actin (phalloidin). Scale bar, 8.5 μm.
    Figure Legend Snippet: Localization of actin isoforms within cultured neurons. Cortical neurons cultured for 4 d were double-labeled with an isoform-specific antibody to β-actin or γ-actin ( left column ) and phalloidin–TRITC ( right column ). A , B , γ-Actin was distributed throughout the cell body ( arrow ) and neurites and resembled phalloidin staining. C , D , Localization of β-actin at tips of minor neurites ( arrowhead ). Low levels of β-actin were observed in the cell body ( arrow ). Phalloidin labeled actin filaments throughout the cell body ( arrow ) and minor neurites ( arrowhead ). E , F , γ-Actin was distributed throughout the axon and growth cone, as was phalloidin staining. G , H , β-Actin was enriched within axonal growth cones ( arrows ). Only weak labeling was observed in the axon shaft. Not all filopodia were labeled ( arrowheads ) despite the presence of F-actin (phalloidin). Scale bar, 8.5 μm.

    Techniques Used: Cell Culture, Labeling, Staining

    Colocalization of β-actin mRNA with microtubules. β-Actin mRNA was detected with rhodamine, and tubulin protein was detected with fluorescein; then the two processed images were superimposed. Pixels that contained both fluorochromes appeared white in optical sections, whereas red pixels denote probe that is not within the same pixel as anti-tubulin ( green pixels ). The majority of β-actin mRNA granules colocalized with microtubules ( white pixels ). Scale bar, 5 μm.
    Figure Legend Snippet: Colocalization of β-actin mRNA with microtubules. β-Actin mRNA was detected with rhodamine, and tubulin protein was detected with fluorescein; then the two processed images were superimposed. Pixels that contained both fluorochromes appeared white in optical sections, whereas red pixels denote probe that is not within the same pixel as anti-tubulin ( green pixels ). The majority of β-actin mRNA granules colocalized with microtubules ( white pixels ). Scale bar, 5 μm.

    Techniques Used:

    Intraneuronal distribution of β-actin and γ-actin mRNA. Cortical neurons were cultured for 4 d, at which time most neurons have distinguishable axonal and dendritic processes. A , Hybridization of biotinated probes to γ-actin mRNA within the cell body ( arrow ). B , Hybridization of digoxigenin-labeled probes to β-actin mRNA within the cell body ( arrow ). C , Differential interference contrast (DIC) microscopy of the cell body ( arrow ), minor neurites, and a single axon. The axon is considerably longer than the minor neurites and cannot be photographed in entirety at this magnification. Shown here is the initial segment. D , Absence of γ-actin mRNAs from the axonal growth cone ( arrow ). E , Localization of β-actin mRNA within the axonal growth cone ( arrow ). F , DIC image of axonal growth cone ( arrow ) from this axon. The axon in this cell is ∼150 μm in length. Scale bar, 10 μm.
    Figure Legend Snippet: Intraneuronal distribution of β-actin and γ-actin mRNA. Cortical neurons were cultured for 4 d, at which time most neurons have distinguishable axonal and dendritic processes. A , Hybridization of biotinated probes to γ-actin mRNA within the cell body ( arrow ). B , Hybridization of digoxigenin-labeled probes to β-actin mRNA within the cell body ( arrow ). C , Differential interference contrast (DIC) microscopy of the cell body ( arrow ), minor neurites, and a single axon. The axon is considerably longer than the minor neurites and cannot be photographed in entirety at this magnification. Shown here is the initial segment. D , Absence of γ-actin mRNAs from the axonal growth cone ( arrow ). E , Localization of β-actin mRNA within the axonal growth cone ( arrow ). F , DIC image of axonal growth cone ( arrow ) from this axon. The axon in this cell is ∼150 μm in length. Scale bar, 10 μm.

    Techniques Used: Cell Culture, Hybridization, Labeling, Microscopy

    Localization of HA–actin protein. Cortical neurons were transfected with an RSV vector containing β-actin bearing an HA epitope tag. A , Detection of HA–actin within the growth cone of a minor process ( arrow ). B , Differential interference contrast (DIC) optics. C , Detection of HA–actin within an axonal growth cone ( arrow ) near another neuron. D , DIC optics. E , Schematic drawing showing the location of the HA sequences between the coding region and the 3′-UTR. Scale bar, 10 μm.
    Figure Legend Snippet: Localization of HA–actin protein. Cortical neurons were transfected with an RSV vector containing β-actin bearing an HA epitope tag. A , Detection of HA–actin within the growth cone of a minor process ( arrow ). B , Differential interference contrast (DIC) optics. C , Detection of HA–actin within an axonal growth cone ( arrow ) near another neuron. D , DIC optics. E , Schematic drawing showing the location of the HA sequences between the coding region and the 3′-UTR. Scale bar, 10 μm.

    Techniques Used: Transfection, Plasmid Preparation

    33) Product Images from "Selective Activation of Sphingosine 1-Phosphate Receptors 1 and 3 Promotes Local Microvascular Network Growth"

    Article Title: Selective Activation of Sphingosine 1-Phosphate Receptors 1 and 3 Promotes Local Microvascular Network Growth

    Journal: Tissue Engineering. Part A

    doi: 10.1089/ten.tea.2010.0404

    S1P receptor-selective activation increases the number of smooth muscle α-actin (SMA)+microvessels. Representative images of SMA-stained sections of dorsal skinfold window chamber tissue after 7 days of treatment with unloaded (A) PLAGA, (B) S1P,
    Figure Legend Snippet: S1P receptor-selective activation increases the number of smooth muscle α-actin (SMA)+microvessels. Representative images of SMA-stained sections of dorsal skinfold window chamber tissue after 7 days of treatment with unloaded (A) PLAGA, (B) S1P,

    Techniques Used: Activation Assay, Staining

    34) Product Images from "Neuronal NLRP1 inflammasome activation of Caspase-1 coordinately regulates inflammatory interleukin-1-beta production and axonal degeneration-associated Caspase-6 activation"

    Article Title: Neuronal NLRP1 inflammasome activation of Caspase-1 coordinately regulates inflammatory interleukin-1-beta production and axonal degeneration-associated Caspase-6 activation

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2015.16

    NLRP1 and Casp1 activate Casp6 in serum-deprived and BzATP-treated neurons. Casp6 VEIDase activity ( a and d ) , TubΔCasp6, full-length tubulin (FL Tub), β -actin western blots ( b and e ) and IL-1 β production ( c and f ) in serum-deprived
    Figure Legend Snippet: NLRP1 and Casp1 activate Casp6 in serum-deprived and BzATP-treated neurons. Casp6 VEIDase activity ( a and d ) , TubΔCasp6, full-length tubulin (FL Tub), β -actin western blots ( b and e ) and IL-1 β production ( c and f ) in serum-deprived

    Techniques Used: Activity Assay, Western Blot

    35) Product Images from "Thymosin α1 Activates Complement Receptor-Mediated Phagocytosis in Human Monocyte-Derived Macrophages"

    Article Title: Thymosin α1 Activates Complement Receptor-Mediated Phagocytosis in Human Monocyte-Derived Macrophages

    Journal: Journal of Innate Immunity

    doi: 10.1159/000351587

    Effect of Tα1 on cytoskeletal components of human MDMs by CLSM. a Images showing the organization of actin and tubulin in Tα1- or LPS-stimulated MDMs. Cells were double stained for F-actin using TRITC-phalloidin (red hue) and for tubulin using the specific monoclonal antibody (green hue). Bars = 25 µm. b , c Recruitment of vinculin and F-actin to the periphagosomal cytoplasm. b Association of vinculin with conidium-internalizing phagosomes in Tα1- or LPS-stimulated MDMs 1 h after challenge. Both cells and conidial morphology were visualized in a bright field modality (BF). c Vinculin and actin assembly on phagosome membrane in Tα1-stimulated MDMs. Cells were double stained for F-actin using TRITC-phalloidin (red hue) and for vinculin using the specific monoclonal antibody (green hue); arrows point to vinculin and actin colocalization into focal structures around the membrane of Tα1-induced phagosome. Bars = 5 µm. (Colors are available in the online version only.)
    Figure Legend Snippet: Effect of Tα1 on cytoskeletal components of human MDMs by CLSM. a Images showing the organization of actin and tubulin in Tα1- or LPS-stimulated MDMs. Cells were double stained for F-actin using TRITC-phalloidin (red hue) and for tubulin using the specific monoclonal antibody (green hue). Bars = 25 µm. b , c Recruitment of vinculin and F-actin to the periphagosomal cytoplasm. b Association of vinculin with conidium-internalizing phagosomes in Tα1- or LPS-stimulated MDMs 1 h after challenge. Both cells and conidial morphology were visualized in a bright field modality (BF). c Vinculin and actin assembly on phagosome membrane in Tα1-stimulated MDMs. Cells were double stained for F-actin using TRITC-phalloidin (red hue) and for vinculin using the specific monoclonal antibody (green hue); arrows point to vinculin and actin colocalization into focal structures around the membrane of Tα1-induced phagosome. Bars = 5 µm. (Colors are available in the online version only.)

    Techniques Used: Confocal Laser Scanning Microscopy, Staining

    36) Product Images from "FAK activation is required for IGF1R-mediated regulation of EMT, migration, and invasion in mesenchymal triple negative breast cancer cells"

    Article Title: FAK activation is required for IGF1R-mediated regulation of EMT, migration, and invasion in mesenchymal triple negative breast cancer cells

    Journal: Oncotarget

    doi:

    Effects of FAK siRNA silencing on IGF1R expression, and cell invasion, proliferation, and survival (A) Western blot analysis of FAK, pIGF1R, and total IGF1R protein levels in MDA-MB-231 and BT549 cells transiently transfected for 48 h with 50 nM of control siRNA, FAK siRNA-1, or FAK siRNA-2. β-actin was used as a loading control. The protein levels were confirmed in three independent experiments. (B) Quantified data of pIGF1R and total IGF1R protein levels, normalized to β-actin. Means and SDs of three separate experiments are shown. (C) Representative images of Transwell cell invasion assays of MDA-MB-231 and BT549 cells transiently transfected with 50 nM of control siRNA, FAK siRNA-1, or FAK siRNA-2. Pictures were taken at 20x magnification. The histograms show the average number of invasive cells (error bars represent S.D. of three independent experiments, each performed in replicates of five. ** p
    Figure Legend Snippet: Effects of FAK siRNA silencing on IGF1R expression, and cell invasion, proliferation, and survival (A) Western blot analysis of FAK, pIGF1R, and total IGF1R protein levels in MDA-MB-231 and BT549 cells transiently transfected for 48 h with 50 nM of control siRNA, FAK siRNA-1, or FAK siRNA-2. β-actin was used as a loading control. The protein levels were confirmed in three independent experiments. (B) Quantified data of pIGF1R and total IGF1R protein levels, normalized to β-actin. Means and SDs of three separate experiments are shown. (C) Representative images of Transwell cell invasion assays of MDA-MB-231 and BT549 cells transiently transfected with 50 nM of control siRNA, FAK siRNA-1, or FAK siRNA-2. Pictures were taken at 20x magnification. The histograms show the average number of invasive cells (error bars represent S.D. of three independent experiments, each performed in replicates of five. ** p

    Techniques Used: Expressing, Western Blot, Multiple Displacement Amplification, Transfection

    Effects of FAK-specific inhibitors on IGF1R activity, invasion, and EMT-related protein expression in TNBC cells (A) Chemical structures of two FAK tyrosine kinase inhibitors, PF-573228 and PF-04554878. The expression of pFAK, FAK, pIGF1R, and IGF1R were assessed via immunoblotting analysis in (B) MDA-MB-231 and (C) BT549 TNBC cells following treatments with indicated doses of PF228 and PF878 for 24 h. (D) Vimentin and E-cadherin expressions examined via Western blotting following treatments as indicated above. β-actin served as a loading control. (E) MDA-MB-231 and BT549 cells were treated with 0.5 μM PF228 or 0.5 μM PF878 for 24 h. Cells were counted by trypan blue exclusion; data represented as a percentage of the vehicle treated (DMSO) control groups. The results represent the average of triplicated treatment groups performed at least three times with reproducible results. (F) Cell lines were treated with 0.5 μM PF228 or 0.5 μM PF878 for 24 h and the effects on cell survival was measured by SRB assays. The data represent mean growth inhibition compared to vehicle treated (DMSO) control cells for three independent experiments for each cell line.
    Figure Legend Snippet: Effects of FAK-specific inhibitors on IGF1R activity, invasion, and EMT-related protein expression in TNBC cells (A) Chemical structures of two FAK tyrosine kinase inhibitors, PF-573228 and PF-04554878. The expression of pFAK, FAK, pIGF1R, and IGF1R were assessed via immunoblotting analysis in (B) MDA-MB-231 and (C) BT549 TNBC cells following treatments with indicated doses of PF228 and PF878 for 24 h. (D) Vimentin and E-cadherin expressions examined via Western blotting following treatments as indicated above. β-actin served as a loading control. (E) MDA-MB-231 and BT549 cells were treated with 0.5 μM PF228 or 0.5 μM PF878 for 24 h. Cells were counted by trypan blue exclusion; data represented as a percentage of the vehicle treated (DMSO) control groups. The results represent the average of triplicated treatment groups performed at least three times with reproducible results. (F) Cell lines were treated with 0.5 μM PF228 or 0.5 μM PF878 for 24 h and the effects on cell survival was measured by SRB assays. The data represent mean growth inhibition compared to vehicle treated (DMSO) control cells for three independent experiments for each cell line.

    Techniques Used: Activity Assay, Expressing, Multiple Displacement Amplification, Western Blot, Sulforhodamine B Assay, Inhibition

    Inhibition of FAK abrogates IGF1R-mediated colony formation, migration, and invasion in TNBC cells (A) Lysates of Hs578T TNBC cells stably expressing empty vector (EV) control or IGF1R-β expression plasmid (IGF1R +/+) were immunoblotted with specific antibodies for IGF1R-β, p-FAK, Vimentin, ZEB-1, E-cadherin, ZO-1, and β-actin loading control. (B) Relative mRNA expression levels of Vimentin and E-cadherin in EV and IGF1R(+/+) TNBC cell lines were detected as described above. Data are displayed in means ± S.D. of at least three independent experiments of each group. (C) Colony formation, (D) spheroid migration, and (E) Matrigel invasion assays were performed as described above on Hs578T TNBC cells expressing either empty vector (EV) control or overexpressing full-length IGF1R-β (IGF1R +/+) followed by treatments with DMSO (control), 0.5 μM PF228, or 0.5 μM PF878 for 24 h. *** p
    Figure Legend Snippet: Inhibition of FAK abrogates IGF1R-mediated colony formation, migration, and invasion in TNBC cells (A) Lysates of Hs578T TNBC cells stably expressing empty vector (EV) control or IGF1R-β expression plasmid (IGF1R +/+) were immunoblotted with specific antibodies for IGF1R-β, p-FAK, Vimentin, ZEB-1, E-cadherin, ZO-1, and β-actin loading control. (B) Relative mRNA expression levels of Vimentin and E-cadherin in EV and IGF1R(+/+) TNBC cell lines were detected as described above. Data are displayed in means ± S.D. of at least three independent experiments of each group. (C) Colony formation, (D) spheroid migration, and (E) Matrigel invasion assays were performed as described above on Hs578T TNBC cells expressing either empty vector (EV) control or overexpressing full-length IGF1R-β (IGF1R +/+) followed by treatments with DMSO (control), 0.5 μM PF228, or 0.5 μM PF878 for 24 h. *** p

    Techniques Used: Inhibition, Migration, Stable Transfection, Expressing, Plasmid Preparation

    Stable silencing of IGF1R confers epithelial-like phenotypes in mesenchymal TNBC cells (A) Endogenous expression of IGF1R-β and total FAK analyzed via Western blot analysis in a panel of mesenchymal human TNBC cells. (B) Western blot confirmation of stable lentiviral knockdown of IGF1R-α/β (IGF1R-KD) in MDA-MB-231 and BT549 TNBC cells. β-actin was used as a loading control. (C) Morphological changes in MDA-MB-231 and BT549 IGF1R-KD cells compared to EV control cells four to six passages post-lentiviral infections; brightfield magnification x20. (D) Western blot analyses of mesenchymal markers (vimentin, Snail-1, ZEB-1), motility marker pFAK, and epithelial markers (E-cadherin, claudin-1, and ZO-1) in cell lines stably expressing EV control plasmid or IGF1R-KD lentiviral plasmids using specific antibodies. β-actin was used as a loading control. (E) Relative mRNA expression levels of Vimentin, ZEB-1, and E-cadherin in MDA-MB-231 EV and IGF1R-KD cell lines was detected by TaqMan quantitative RT-PCR and normalized to RPLPO. The relative amounts of transcript were described using the 2–ΔΔCt method. Data are displayed in means ± standard deviation of at least three independent experiments of each group.
    Figure Legend Snippet: Stable silencing of IGF1R confers epithelial-like phenotypes in mesenchymal TNBC cells (A) Endogenous expression of IGF1R-β and total FAK analyzed via Western blot analysis in a panel of mesenchymal human TNBC cells. (B) Western blot confirmation of stable lentiviral knockdown of IGF1R-α/β (IGF1R-KD) in MDA-MB-231 and BT549 TNBC cells. β-actin was used as a loading control. (C) Morphological changes in MDA-MB-231 and BT549 IGF1R-KD cells compared to EV control cells four to six passages post-lentiviral infections; brightfield magnification x20. (D) Western blot analyses of mesenchymal markers (vimentin, Snail-1, ZEB-1), motility marker pFAK, and epithelial markers (E-cadherin, claudin-1, and ZO-1) in cell lines stably expressing EV control plasmid or IGF1R-KD lentiviral plasmids using specific antibodies. β-actin was used as a loading control. (E) Relative mRNA expression levels of Vimentin, ZEB-1, and E-cadherin in MDA-MB-231 EV and IGF1R-KD cell lines was detected by TaqMan quantitative RT-PCR and normalized to RPLPO. The relative amounts of transcript were described using the 2–ΔΔCt method. Data are displayed in means ± standard deviation of at least three independent experiments of each group.

    Techniques Used: Expressing, Western Blot, Multiple Displacement Amplification, Marker, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation

    37) Product Images from "LRRK2 deficiency impairs trans-Golgi to lysosome trafficking and endocytic cargo degradation in human renal proximal tubule epithelial cells"

    Article Title: LRRK2 deficiency impairs trans-Golgi to lysosome trafficking and endocytic cargo degradation in human renal proximal tubule epithelial cells

    Journal: American Journal of Physiology - Renal Physiology

    doi: 10.1152/ajprenal.00009.2018

    LRRK2 and NSF physically and functionally interact in renal epithelia. A : confocal immunofluorescent images of HK2 cells after transfection with FLAG-LRRK2 expression vectors to enable visualization of LRRK2 (red) with endogenous NSF (green). The inset in the merged panel is a ×2.5 magnification of the indicated cell in this image. B : quantification of immunofluorescent signal colocalization between total LRRK2 and the FLAG epitope found on exogenous LRRK2 (positive control), and between NSF and the FLAG epitope using Pearson’s and Mander’s correlation methods. Data represent mean signal overlap from 200 cells for each condition imaged by confocal microscopy. Error bars represent standard deviations of these measurements. C : immunoblot of NSF and LRRK2 after endogenous coimmunoprecipitation with anti-LRRK2 antibodies. Cell lysis buffer with and without 2 mM N -ethylmaleimide (NEM) was used to harvest protein lysates from cells. Input samples represent 10% of the protein input used for immunoprecipitations. D : relative expression of NSF at the mRNA and protein levels in control, LRRK2, or NSF knockdown cell lines. Expression was determined as the ratio of NSF to RPL13A mRNA (qRT-PCR) or β-actin protein (immunoblot) and then normalized to expression in nontargeting control cells (NT-sh). Error bars indicate standard deviations of triplicate samples. E : lysates were harvested from parental HK2 cells (control) and cells stably transfected with V5-tagged human NSF (nV5-NSF) after each line was infected with the indicated lentiviral shRNA vectors. Representative immunoblots demonstrate NSF and V5-tag expression levels relative to actin in cells after introduction of shRNAs. F : immunoblot analysis of lysates from HK2 cells after infection with BacMam/FLAG-LRRK2 at various multiplicities of infection (shown as percent by volume of viral suspension used). Increasing LRRK2 levels has no impact on total levels of NSF protein in cells. Tubulin was used as a loading control to indicate equal loading between samples. LRRK2, leucine-rich repeat kinase 2; HK2, normal human kidney cells; NSF, N -ethylmaleimide-sensitive fusion protein; qRT-PCR, quantitative RT-PCR; shRNA, short hairpin RNA.
    Figure Legend Snippet: LRRK2 and NSF physically and functionally interact in renal epithelia. A : confocal immunofluorescent images of HK2 cells after transfection with FLAG-LRRK2 expression vectors to enable visualization of LRRK2 (red) with endogenous NSF (green). The inset in the merged panel is a ×2.5 magnification of the indicated cell in this image. B : quantification of immunofluorescent signal colocalization between total LRRK2 and the FLAG epitope found on exogenous LRRK2 (positive control), and between NSF and the FLAG epitope using Pearson’s and Mander’s correlation methods. Data represent mean signal overlap from 200 cells for each condition imaged by confocal microscopy. Error bars represent standard deviations of these measurements. C : immunoblot of NSF and LRRK2 after endogenous coimmunoprecipitation with anti-LRRK2 antibodies. Cell lysis buffer with and without 2 mM N -ethylmaleimide (NEM) was used to harvest protein lysates from cells. Input samples represent 10% of the protein input used for immunoprecipitations. D : relative expression of NSF at the mRNA and protein levels in control, LRRK2, or NSF knockdown cell lines. Expression was determined as the ratio of NSF to RPL13A mRNA (qRT-PCR) or β-actin protein (immunoblot) and then normalized to expression in nontargeting control cells (NT-sh). Error bars indicate standard deviations of triplicate samples. E : lysates were harvested from parental HK2 cells (control) and cells stably transfected with V5-tagged human NSF (nV5-NSF) after each line was infected with the indicated lentiviral shRNA vectors. Representative immunoblots demonstrate NSF and V5-tag expression levels relative to actin in cells after introduction of shRNAs. F : immunoblot analysis of lysates from HK2 cells after infection with BacMam/FLAG-LRRK2 at various multiplicities of infection (shown as percent by volume of viral suspension used). Increasing LRRK2 levels has no impact on total levels of NSF protein in cells. Tubulin was used as a loading control to indicate equal loading between samples. LRRK2, leucine-rich repeat kinase 2; HK2, normal human kidney cells; NSF, N -ethylmaleimide-sensitive fusion protein; qRT-PCR, quantitative RT-PCR; shRNA, short hairpin RNA.

    Techniques Used: Transfection, Expressing, FLAG-tag, Positive Control, Confocal Microscopy, Lysis, Quantitative RT-PCR, Western Blot, Stable Transfection, Infection, shRNA

    38) Product Images from "Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells"

    Article Title: Effect of overexpression of β- and γ-actin isoforms on actin cytoskeleton organization and migration of human colon cancer cells

    Journal: Histochemistry and Cell Biology

    doi: 10.1007/s00418-014-1199-9

    Wound healing assay of BE cells overexpressing β- or γ-actin. a Shows representative experiments, where photographs were taken 24 h after scratching of the cell monolayer. Scale bar 50 μm. Diagram b shows migration velocity of BE cell variants. Asterisk indicates value statistically different from those obtained for the control, transfected with pAcGFP-C1 plasmid cells. The significance level was set at P ≤ 0.05 in Student’s t test
    Figure Legend Snippet: Wound healing assay of BE cells overexpressing β- or γ-actin. a Shows representative experiments, where photographs were taken 24 h after scratching of the cell monolayer. Scale bar 50 μm. Diagram b shows migration velocity of BE cell variants. Asterisk indicates value statistically different from those obtained for the control, transfected with pAcGFP-C1 plasmid cells. The significance level was set at P ≤ 0.05 in Student’s t test

    Techniques Used: Wound Healing Assay, Migration, Transfection, Plasmid Preparation

    Changes in actin polymerization state in the BE cells overexpressing β- or γ-actin. Results were compared to cells transfected with an empty vector pAcGFP-C1. The data were obtained from three independent experiments
    Figure Legend Snippet: Changes in actin polymerization state in the BE cells overexpressing β- or γ-actin. Results were compared to cells transfected with an empty vector pAcGFP-C1. The data were obtained from three independent experiments

    Techniques Used: Transfection, Plasmid Preparation

    Subcellular distribution of β- ( a ) and γ-actin ( b ) in examined cells overexpressing actin isoforms. Lower rows in a and b shows representative BE cells overexpressing β- or γ-actin, respectively. Left panel AcGFP fluorescence ( green ), middle panel endogenous β- or γ-actin stained with mouse anti-β- or anti-γ-actin antibody ( red ). Merged images are shown in the right panel . Long arrows show colocalization of AcGFP-actin and endogenous actin in lamellipodia and short ones probably in retracting tail areas. Scale bar 10 μm
    Figure Legend Snippet: Subcellular distribution of β- ( a ) and γ-actin ( b ) in examined cells overexpressing actin isoforms. Lower rows in a and b shows representative BE cells overexpressing β- or γ-actin, respectively. Left panel AcGFP fluorescence ( green ), middle panel endogenous β- or γ-actin stained with mouse anti-β- or anti-γ-actin antibody ( red ). Merged images are shown in the right panel . Long arrows show colocalization of AcGFP-actin and endogenous actin in lamellipodia and short ones probably in retracting tail areas. Scale bar 10 μm

    Techniques Used: Fluorescence, Staining

    Migration ( a ) and invasion ( b ) capacities of BE cells overexpressing β- or γ-actin isoform. Results expressed as the mean ± SD are representative for at least three independent experiments. Migration and invasion in control cells are presented as 100 %. Asterisks indicate values statistically different from those obtained for the control, transfected with pAcGFP-C1 plasmid cells. The significance level was set at P ≤ 0.05 in Student’s t test
    Figure Legend Snippet: Migration ( a ) and invasion ( b ) capacities of BE cells overexpressing β- or γ-actin isoform. Results expressed as the mean ± SD are representative for at least three independent experiments. Migration and invasion in control cells are presented as 100 %. Asterisks indicate values statistically different from those obtained for the control, transfected with pAcGFP-C1 plasmid cells. The significance level was set at P ≤ 0.05 in Student’s t test

    Techniques Used: Migration, Transfection, Plasmid Preparation

    Filamentous actin organization in BE colon cancer cells overexpressing β- and γ-actin isoforms. a Confocal images showing cells expressing actin isoform β or γ encoded by pAcGFP-C1 expression vector were compared to cells transfected with an empty vector pAcGFP-C1. Left panel AcGFP fluorescence ( green ). Middle panel filamentous actin visualized by staining with AlexaFluor ® 568-conjugated phalloidin ( red ). Merged images are shown in the right panel. Scale bar 10 μm. Arrows indicate the areas of colocalization of presumed overexpressed β- or γ-actin with F actin. b Upper row— confocal images showing area of lamellipodial protrusions in all transfected cells (enlarged fragments from merged images from Fig. 5 a, rectangles squares ). Lower row —actin-rich protrusions areas. The data were counted for 20 cells in each case. Asterisks indicate values statistically different from those obtained for the control cells. The significance level was set at P ≤ 0.05 in Student’s t test
    Figure Legend Snippet: Filamentous actin organization in BE colon cancer cells overexpressing β- and γ-actin isoforms. a Confocal images showing cells expressing actin isoform β or γ encoded by pAcGFP-C1 expression vector were compared to cells transfected with an empty vector pAcGFP-C1. Left panel AcGFP fluorescence ( green ). Middle panel filamentous actin visualized by staining with AlexaFluor ® 568-conjugated phalloidin ( red ). Merged images are shown in the right panel. Scale bar 10 μm. Arrows indicate the areas of colocalization of presumed overexpressed β- or γ-actin with F actin. b Upper row— confocal images showing area of lamellipodial protrusions in all transfected cells (enlarged fragments from merged images from Fig. 5 a, rectangles squares ). Lower row —actin-rich protrusions areas. The data were counted for 20 cells in each case. Asterisks indicate values statistically different from those obtained for the control cells. The significance level was set at P ≤ 0.05 in Student’s t test

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Fluorescence, Staining

    Subcellular localization of monomeric actin in cells overexpressing β- or γ-actin. β- or γ-actin was encoded by pAcGFP-C1 expression vector. Left panel AcGFP ( green ). Middle panel monomeric actin visualized by staining with DNase I conjugated with Alexa Fluor ® 594 ( red ). Merged images are shown in the right panel . Scale bar 10 μm
    Figure Legend Snippet: Subcellular localization of monomeric actin in cells overexpressing β- or γ-actin. β- or γ-actin was encoded by pAcGFP-C1 expression vector. Left panel AcGFP ( green ). Middle panel monomeric actin visualized by staining with DNase I conjugated with Alexa Fluor ® 594 ( red ). Merged images are shown in the right panel . Scale bar 10 μm

    Techniques Used: Expressing, Plasmid Preparation, Staining

    Molecular biological strategy for qRT-PCR analysis. a A schematic map of a part of pAcGFP-C1-actin vector, CMV—cytomegalovirus promoter. Sites to which specific primers anneal are shown as bold bars . b Control PCR reactions after RT-PCR on mRNA isolated from BE cells proving there were no contaminations while performing qRT-PCR analysis. Primers recognizing cDNA of β-actin were used, expected band size: 174 nt. c Control PCR reactions where pOTB7 plasmids with clones of β- and γ-actin and cDNA of BE cells served as templates; for this reaction, primers for subcloning of isoactins into pAcGFP-C1 were used. Expected band size 1,724 nt. d Control PCR reactions, where cDNAs of control and transfected cells served as templates and those recognizing cDNA of AcGFP-actins were taken as primers (β for AcGFP-β-actin, γ for AcGFP-γ-actin). Expected band sizes: 222 nt
    Figure Legend Snippet: Molecular biological strategy for qRT-PCR analysis. a A schematic map of a part of pAcGFP-C1-actin vector, CMV—cytomegalovirus promoter. Sites to which specific primers anneal are shown as bold bars . b Control PCR reactions after RT-PCR on mRNA isolated from BE cells proving there were no contaminations while performing qRT-PCR analysis. Primers recognizing cDNA of β-actin were used, expected band size: 174 nt. c Control PCR reactions where pOTB7 plasmids with clones of β- and γ-actin and cDNA of BE cells served as templates; for this reaction, primers for subcloning of isoactins into pAcGFP-C1 were used. Expected band size 1,724 nt. d Control PCR reactions, where cDNAs of control and transfected cells served as templates and those recognizing cDNA of AcGFP-actins were taken as primers (β for AcGFP-β-actin, γ for AcGFP-γ-actin). Expected band sizes: 222 nt

    Techniques Used: Quantitative RT-PCR, Plasmid Preparation, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Isolation, Clone Assay, Subcloning, Transfection

    mRNA levels of actin isoforms in cells transiently transfected with plasmids coding for β- or γ-actin. The mRNA levels of AcGFP-β-actin ( a ) or AcGFP-γ-actin ( b ) and total β- ( c ) or γ-actin ( d ) were measured by qRT-PCR. On pictures c and d , we showed content of endogenous ( dark gray ) and exogenous ( light gray ) mRNA of actin in total actin mRNA level. Asterisks indicate values statistically different from those obtained for the control cells, transfected with pAcGFP-C1 plasmid. The significance level was set at P ≤ 0.05 in Student’s t test
    Figure Legend Snippet: mRNA levels of actin isoforms in cells transiently transfected with plasmids coding for β- or γ-actin. The mRNA levels of AcGFP-β-actin ( a ) or AcGFP-γ-actin ( b ) and total β- ( c ) or γ-actin ( d ) were measured by qRT-PCR. On pictures c and d , we showed content of endogenous ( dark gray ) and exogenous ( light gray ) mRNA of actin in total actin mRNA level. Asterisks indicate values statistically different from those obtained for the control cells, transfected with pAcGFP-C1 plasmid. The significance level was set at P ≤ 0.05 in Student’s t test

    Techniques Used: Transfection, Quantitative RT-PCR, Plasmid Preparation

    Subcellular organization of β- and γ-actins as well as filamentous and monomeric actin in non-transfected BE cells. Left picture β-actin stained with mouse monoclonal antibodies directed against β-actin. Second picture γ-actin stained with mouse monoclonal antibodies directed against γ-actin. Third picture filamentous actin visualized by staining with AlexaFluor ® 568-conjugated phalloidin. Right picture monomeric actin visualized by staining with DNase I conjugated with Alexa Fluor ® 594. Long arrows show localization of actin within lamellipodia and short arrows point at invadopodia. Scale bar 20 μm
    Figure Legend Snippet: Subcellular organization of β- and γ-actins as well as filamentous and monomeric actin in non-transfected BE cells. Left picture β-actin stained with mouse monoclonal antibodies directed against β-actin. Second picture γ-actin stained with mouse monoclonal antibodies directed against γ-actin. Third picture filamentous actin visualized by staining with AlexaFluor ® 568-conjugated phalloidin. Right picture monomeric actin visualized by staining with DNase I conjugated with Alexa Fluor ® 594. Long arrows show localization of actin within lamellipodia and short arrows point at invadopodia. Scale bar 20 μm

    Techniques Used: Transfection, Staining

    Western blot analysis of AcGFP, β-actin and γ-actin. A representative immunoblots identificating AcGFP and fusion proteins ( a ) as well as β-actin ( b ) and γ-actin ( c ) in cellular extracts of control cells (transfected with pAcGFP-C1) and cells overexpressing AcGFP tagged β- or γ-actin. Used antibodies: mouse monoclonal antibodies directed against β tubulin, mouse monoclonal antibodies directed against GFP, mouse monoclonal anti-β-actin antibodies and mouse monoclonal anti-γ-actin antibodies
    Figure Legend Snippet: Western blot analysis of AcGFP, β-actin and γ-actin. A representative immunoblots identificating AcGFP and fusion proteins ( a ) as well as β-actin ( b ) and γ-actin ( c ) in cellular extracts of control cells (transfected with pAcGFP-C1) and cells overexpressing AcGFP tagged β- or γ-actin. Used antibodies: mouse monoclonal antibodies directed against β tubulin, mouse monoclonal antibodies directed against GFP, mouse monoclonal anti-β-actin antibodies and mouse monoclonal anti-γ-actin antibodies

    Techniques Used: Western Blot, Transfection

    39) Product Images from "Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes"

    Article Title: Cortical Granule Exocytosis Is Mediated by Alpha-SNAP and N-Ethilmaleimide Sensitive Factor in Mouse Oocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135679

    Detection of α-SNAP, γ-SNAP and NSF by Western blot. A .  Left ,  Upper panels : Inmunoblot of α-SNAP: Protein extracts from equal numbers (150) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10mM strontium chloride (SrCl 2 ) were separated on a 12% SDS-PAGE gel. Positive controls: mouse brain (Brain, 1.25 μg) and recombinant His6-α-SNAP (α SNAP rec, 5 ng). Immunoblot of β- tubulin (β Tub) was performed as a control of protein loading.  Lower panels : Immunoblot using anti-α-SNAP antibody preabsorbed with full lenght α-SNAP recombinant protein (α SNAP pb).  Right , densitometry analysis of Western blots for α-SNAP (mean ± SEM, n = 4) showing α-SNAP protein expression level (α SNAP/β Tub ratio) relative to GV expression, set as 1.  B .  Left ,  Upper panels : Inmunoblot of γ-SNAP: Protein extracts from equal numbers (300) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10mM strontium chloride (SrCl 2 ) were separated on a 12% SDS-PAGE gel. Positive controls: mouse brain (Brain, 6 μg) and recombinant thrombine cleaved γ-SNAP-GST (γ SNAP rec, 0.2 μg). Immunoblot of β- tubulin (β Tub) was performed as a control of protein loading.  Lower panels : Immunoblot using anti-γ-SNAP antibody preabsorbed with γ-SNAP control peptide (γ SNAP pb).  Right , densitometry analysis of Western blots for γ-SNAP (mean ± SEM, n = 3) showing γ-SNAP protein expression level (γ SNAP/β Tub ratio) relative to GV expression, set as 1.  C .  Left ,  Upper panels : Inmunoblot of NSF: Protein extracts from equal numbers (200) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10 mM strontium chloride (SrCl 2 ) were separated on a 15% SDS-PAGE gel. Positive controls: mouse brain (Brain, 3,5 μg) and recombinant His6-NSF (NSF rec, 75 ng). Immunoblot of β- actin (β Act) was performed as a control of protein loading.  Lower panels : Immunoblot using anti-NSF antibody preabsorbed with NSF control peptide (NSF pb).  Right , densitometry analysis of Western blots for NSF (mean ± SEM, n = 3) showing NSF protein expression level (NSF/ β Act ratio) relative to GV expression, set as 1. In all panels MW protein standards (x10 3 ) are indicated on the right and primary antibodies, on the left.
    Figure Legend Snippet: Detection of α-SNAP, γ-SNAP and NSF by Western blot. A . Left , Upper panels : Inmunoblot of α-SNAP: Protein extracts from equal numbers (150) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10mM strontium chloride (SrCl 2 ) were separated on a 12% SDS-PAGE gel. Positive controls: mouse brain (Brain, 1.25 μg) and recombinant His6-α-SNAP (α SNAP rec, 5 ng). Immunoblot of β- tubulin (β Tub) was performed as a control of protein loading. Lower panels : Immunoblot using anti-α-SNAP antibody preabsorbed with full lenght α-SNAP recombinant protein (α SNAP pb). Right , densitometry analysis of Western blots for α-SNAP (mean ± SEM, n = 4) showing α-SNAP protein expression level (α SNAP/β Tub ratio) relative to GV expression, set as 1. B . Left , Upper panels : Inmunoblot of γ-SNAP: Protein extracts from equal numbers (300) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10mM strontium chloride (SrCl 2 ) were separated on a 12% SDS-PAGE gel. Positive controls: mouse brain (Brain, 6 μg) and recombinant thrombine cleaved γ-SNAP-GST (γ SNAP rec, 0.2 μg). Immunoblot of β- tubulin (β Tub) was performed as a control of protein loading. Lower panels : Immunoblot using anti-γ-SNAP antibody preabsorbed with γ-SNAP control peptide (γ SNAP pb). Right , densitometry analysis of Western blots for γ-SNAP (mean ± SEM, n = 3) showing γ-SNAP protein expression level (γ SNAP/β Tub ratio) relative to GV expression, set as 1. C . Left , Upper panels : Inmunoblot of NSF: Protein extracts from equal numbers (200) of GV-intact oocytes (GV), MII oocytes (MII) and parthenogenetic activated MII oocytes with 10 mM strontium chloride (SrCl 2 ) were separated on a 15% SDS-PAGE gel. Positive controls: mouse brain (Brain, 3,5 μg) and recombinant His6-NSF (NSF rec, 75 ng). Immunoblot of β- actin (β Act) was performed as a control of protein loading. Lower panels : Immunoblot using anti-NSF antibody preabsorbed with NSF control peptide (NSF pb). Right , densitometry analysis of Western blots for NSF (mean ± SEM, n = 3) showing NSF protein expression level (NSF/ β Act ratio) relative to GV expression, set as 1. In all panels MW protein standards (x10 3 ) are indicated on the right and primary antibodies, on the left.

    Techniques Used: Western Blot, SDS Page, Recombinant, Expressing, Activated Clotting Time Assay

    40) Product Images from "Cell cycle-dependent regulation of the RNA-binding protein Staufen1"

    Article Title: Cell cycle-dependent regulation of the RNA-binding protein Staufen1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku506

    Stau1 protein levels vary during the cell cycle. ( A ) HCT116 cells were grown asynchronously (AS) or were synchronized at the G 1 /S transition by a double thymidine block (DTB) and then released by addition of fresh medium. Cell extracts were prepared at different time points post-release as indicated and analyzed by western blotting (top and middle panels). CyclinB, marker of S, G 2 and M phases; AuroraA, marker of G 2 and M phases; MPM2, mitosis protein monoclonal 2 detects a variety of phosphorylated proteins during mitosis. DNA content was assessed by flow cytometry analysis at the indicated time points after release from thymidine block (bottom panel). ( B ) HCT116 cells blocked in prometaphase with nocodazole (NB) were collected by shake-off and replated in fresh medium. Cell extracts were prepared at different time points and analyzed by western blotting. (Bottom) RNA was isolated from the cell extracts used for western blot analysis and the levels of mRNA were quantified by RT-qPCR. The ratios between Stau1 and GAPDH mRNA levels were calculated at each time point and the means were plotted relative to that obtained at time 0, which was arbitrarily set to 1. ( C ) U2OS cells were grown asynchronously (As) or were synchronized at the G 1 /S transition (DTB), in prometaphase (NB) or in G 1 by a nocodazole block followed by a release of 3 h in fresh medium (NB + 3 h). Stau1 levels were monitored by western blotting. Cyclin A, marker of the G 1 /S transition. Western blot results (A, B and C) are representatives of three independently performed experiments that showed similar profiles. Statistical analyses: Quantification of the relative amounts of Stau1 protein and/or mRNA at each time point, expressed as the mean of three independent experiments. Standard deviations are shown and statistical analyses (Student's t -test) are indicated when significant. For protein analysis, the ratios between Stau1 and β-Actin levels were calculated at each time point and the means were plotted relative to that obtained at time 0 (A and B) or DTB (C), which was arbitrarily set to 1. Quantifications of cyclin B1, Aurora A and MPM2 are provided in Supplementary Figure S1.
    Figure Legend Snippet: Stau1 protein levels vary during the cell cycle. ( A ) HCT116 cells were grown asynchronously (AS) or were synchronized at the G 1 /S transition by a double thymidine block (DTB) and then released by addition of fresh medium. Cell extracts were prepared at different time points post-release as indicated and analyzed by western blotting (top and middle panels). CyclinB, marker of S, G 2 and M phases; AuroraA, marker of G 2 and M phases; MPM2, mitosis protein monoclonal 2 detects a variety of phosphorylated proteins during mitosis. DNA content was assessed by flow cytometry analysis at the indicated time points after release from thymidine block (bottom panel). ( B ) HCT116 cells blocked in prometaphase with nocodazole (NB) were collected by shake-off and replated in fresh medium. Cell extracts were prepared at different time points and analyzed by western blotting. (Bottom) RNA was isolated from the cell extracts used for western blot analysis and the levels of mRNA were quantified by RT-qPCR. The ratios between Stau1 and GAPDH mRNA levels were calculated at each time point and the means were plotted relative to that obtained at time 0, which was arbitrarily set to 1. ( C ) U2OS cells were grown asynchronously (As) or were synchronized at the G 1 /S transition (DTB), in prometaphase (NB) or in G 1 by a nocodazole block followed by a release of 3 h in fresh medium (NB + 3 h). Stau1 levels were monitored by western blotting. Cyclin A, marker of the G 1 /S transition. Western blot results (A, B and C) are representatives of three independently performed experiments that showed similar profiles. Statistical analyses: Quantification of the relative amounts of Stau1 protein and/or mRNA at each time point, expressed as the mean of three independent experiments. Standard deviations are shown and statistical analyses (Student's t -test) are indicated when significant. For protein analysis, the ratios between Stau1 and β-Actin levels were calculated at each time point and the means were plotted relative to that obtained at time 0 (A and B) or DTB (C), which was arbitrarily set to 1. Quantifications of cyclin B1, Aurora A and MPM2 are provided in Supplementary Figure S1.

    Techniques Used: Blocking Assay, Western Blot, Marker, Flow Cytometry, Cytometry, Isolation, Quantitative RT-PCR

    Stau1 down-regulation in mitosis is dependent on the APC/C. (A) HEK293T cells were co-transfected with plasmids coding for Stau1 55 -FLAG 3 and HA-Cdc20, HA-Cdh1 or the empty vector as control. Cells extracts were analyzed by western blotting. *, unspecific signal. (B) HCT116 cells were transfected with siRNAs control or targeting the Cdc20 mRNA. Cells were synchronized in late G2 with RO-3306 (0) and released from the block for 3 h ( 3 ). Cell extracts were analyzed by western blotting. In A and B, the ratios between Stau1 55 -FLAG and β-Actin levels and the statistical analyses were calculated as in the legend of Figure 1 , the ratio observed in cells transfected with the empty vector being arbitrarily set to 1. (C) HEK293T cells were transfected with plasmids coding for FLAG-Cdh1, FLAG-Cdc20 or FLAG-YFP as indicated. Left: input. Right: FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody and co-purified endogenous Stau1 was detected with anti-Stau1 antibody by western blotting. Each panel (A, B and C) is representative of three independently performed experiments.
    Figure Legend Snippet: Stau1 down-regulation in mitosis is dependent on the APC/C. (A) HEK293T cells were co-transfected with plasmids coding for Stau1 55 -FLAG 3 and HA-Cdc20, HA-Cdh1 or the empty vector as control. Cells extracts were analyzed by western blotting. *, unspecific signal. (B) HCT116 cells were transfected with siRNAs control or targeting the Cdc20 mRNA. Cells were synchronized in late G2 with RO-3306 (0) and released from the block for 3 h ( 3 ). Cell extracts were analyzed by western blotting. In A and B, the ratios between Stau1 55 -FLAG and β-Actin levels and the statistical analyses were calculated as in the legend of Figure 1 , the ratio observed in cells transfected with the empty vector being arbitrarily set to 1. (C) HEK293T cells were transfected with plasmids coding for FLAG-Cdh1, FLAG-Cdc20 or FLAG-YFP as indicated. Left: input. Right: FLAG-tagged proteins were immunoprecipitated with anti-FLAG antibody and co-purified endogenous Stau1 was detected with anti-Stau1 antibody by western blotting. Each panel (A, B and C) is representative of three independently performed experiments.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Blocking Assay, Immunoprecipitation, Purification

    Stau1 is a substrate of the ubiquitin proteasome system. Untransfected HCT116, HEK293T and U2OS cells (A) and Stau1 55 -FLAG-transfected HEK293T cells (B) were treated for 6 h with the proteasome inhibitor MG132 (10 μM) or by DMSO (the MG132 vehicle) as control. Cell extracts were analyzed by western blotting. The ratios between Stau1 and β-Actin levels and the statistical analyses were calculated as described in the legend of Figure 1 , the ratio observed in cells treated with Dimethylsulfoxide (DMSO) being arbitrarily set to 1. (C) HEK293T cells were transfected with plasmids coding for Stau1 55 -HA 3 and/or GFP-Ubiquitin. Left: input. Right: Stau1 55 -HA 3 was immunoprecipitated with a mouse monoclonal anti-HA antibody (12CA5) and co-immunoprecipitated proteins were analyzed by western blotting. *, unspecific signal. Each panel is representative of three independently performed experiments that generated similar results.
    Figure Legend Snippet: Stau1 is a substrate of the ubiquitin proteasome system. Untransfected HCT116, HEK293T and U2OS cells (A) and Stau1 55 -FLAG-transfected HEK293T cells (B) were treated for 6 h with the proteasome inhibitor MG132 (10 μM) or by DMSO (the MG132 vehicle) as control. Cell extracts were analyzed by western blotting. The ratios between Stau1 and β-Actin levels and the statistical analyses were calculated as described in the legend of Figure 1 , the ratio observed in cells treated with Dimethylsulfoxide (DMSO) being arbitrarily set to 1. (C) HEK293T cells were transfected with plasmids coding for Stau1 55 -HA 3 and/or GFP-Ubiquitin. Left: input. Right: Stau1 55 -HA 3 was immunoprecipitated with a mouse monoclonal anti-HA antibody (12CA5) and co-immunoprecipitated proteins were analyzed by western blotting. *, unspecific signal. Each panel is representative of three independently performed experiments that generated similar results.

    Techniques Used: Transfection, Western Blot, Immunoprecipitation, Generated

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    Incubation:

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    Chemiluminescence Immunoassay:

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    Article Snippet: .. The membrane was blocked with 10% non-fat milk in Tris buffered saline containing Tween-20 (TBS-T) (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween-20) and then incubated with primary human antibodies against β-actin (Sigma), Bcl-2, JNK, p-JNK, p-ERK, procaspase-3 (Santa Cruz), caspase-9 (Stressgen), and P-glycoprotein (Merck) in TBS-T. After incubation with the secondary antibody conjugated with horseradish peroxidase, immunodetected proteins were visualized by using an enhanced chemiluminescence assay kit (Amersham Life Science). ..

    SDS Page:

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    Millipore anti α sma
    Preischemic administration of stromal vascular fraction (SVF) inhibited TGF-β1-induced epithelia-mesenchymal transition (EMT) and microvascular rarefaction. (A–C): Relative abundance of E-cadherin/GAPDH (A) and <t>α-SMA/GAPDH</t> (B)
    Anti α Sma, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse anti human β actin monoclonal antibody
    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with <t>anti-β-actin</t> antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.
    Mouse Anti Human β Actin Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monoclonal rabbit anti β actin
    Nerve injury increases protein expressions of Iba1, GFAP, TNF-α, IL-1β, and MCP-1 in the spinal dorsal horn Bar graphs show the mean (+ S.E.) density of Iba1, GFAP, TNF-α, IL-1β, and MCP-1 relative to <t>β-actin</t> in the spinal dorsal horn in rats receiving either sham operation or pSNL on day 3 (A) and day 10 (B) after surgery. Samples of each molecular protein expression in each group are displayed. * p
    Monoclonal Rabbit Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Preischemic administration of stromal vascular fraction (SVF) inhibited TGF-β1-induced epithelia-mesenchymal transition (EMT) and microvascular rarefaction. (A–C): Relative abundance of E-cadherin/GAPDH (A) and α-SMA/GAPDH (B)

    Journal: Stem Cells Translational Medicine

    Article Title: Preischemic Administration of Nonexpanded Adipose Stromal Vascular Fraction Attenuates Acute Renal Ischemia/Reperfusion Injury and Fibrosis

    doi: 10.5966/sctm.2015-0223

    Figure Lengend Snippet: Preischemic administration of stromal vascular fraction (SVF) inhibited TGF-β1-induced epithelia-mesenchymal transition (EMT) and microvascular rarefaction. (A–C): Relative abundance of E-cadherin/GAPDH (A) and α-SMA/GAPDH (B)

    Article Snippet: The membranes were blocked with 10% skimmed milk for 1 hour and incubated at 4°C overnight with anti-α-SMA, anti-E-cadherin, or anti-glyceraldehyde-3-phosphate dehydrogenase (EMD Millipore), followed by incubation with horseradish peroxidase-conjugated anti-mouse IgG (Cell Signaling, Danvers, MA, ).

    Techniques:

    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.

    Journal: PLoS ONE

    Article Title: Ser276 Phosphorylation of NF-kB p65 by MSK1 Controls SCF Expression in Inflammation

    doi: 10.1371/journal.pone.0004393

    Figure Lengend Snippet: Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with anti-β-actin antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.

    Article Snippet: Immunoblotting used the following antibodies: rabbit anti-human IκB-α polyclonal antibody (1/1000, Calbiochem, La Jolla, CA), mouse anti-human phospho- IκB-α monoclonal antibody, (1/1000, Ab-1, Oncogene Research Product, Boston, MA), rabbit anti-human phospho-Ser276 p65 antibody (1/1000, 3037, Cell Signaling Technology, Danvers MA), rabbit anti-human phospho-Ser536 p65 antibody (1/1000, 3031, Cell Signaling Technology), rabbit anti-human p65 polyclonal antibody (1/200, sc-109, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-human CBP polyclonal antibody (1/200, sc-369, Santa Cruz Biotechnology), mouse anti-human β-actin monoclonal antibody (1/5000, Ab-1, Oncogene Research Product), goat anti-human MSK1 (1/200, sc-9392, Santa Cruz Biotechnology.

    Techniques: Expressing, Transfection, Luciferase, Construct, Incubation, Activity Assay, Inhibition, Western Blot, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

    Nerve injury increases protein expressions of Iba1, GFAP, TNF-α, IL-1β, and MCP-1 in the spinal dorsal horn Bar graphs show the mean (+ S.E.) density of Iba1, GFAP, TNF-α, IL-1β, and MCP-1 relative to β-actin in the spinal dorsal horn in rats receiving either sham operation or pSNL on day 3 (A) and day 10 (B) after surgery. Samples of each molecular protein expression in each group are displayed. * p

    Journal: Neuroscience

    Article Title: EZH2 regulates spinal neuroinflammation in rats with neuropathic pain

    doi: 10.1016/j.neuroscience.2017.02.041

    Figure Lengend Snippet: Nerve injury increases protein expressions of Iba1, GFAP, TNF-α, IL-1β, and MCP-1 in the spinal dorsal horn Bar graphs show the mean (+ S.E.) density of Iba1, GFAP, TNF-α, IL-1β, and MCP-1 relative to β-actin in the spinal dorsal horn in rats receiving either sham operation or pSNL on day 3 (A) and day 10 (B) after surgery. Samples of each molecular protein expression in each group are displayed. * p

    Article Snippet: The membranes were blocked with 5% milk or 5% BSA in TBST, and then incubated respectively overnight at 4 °C with polyclonal rabbit anti-GFAP (1:1,000, cell signaling), polyclonal rabbit anti-Iba1 (1:1,000; Wako), rabbit anti-TNF-α (1:500; Millipore), rabbit anti-IL-1β (1:500; Millipore), rabbit anti-MCP-1 (1:500; Abcam), rabbit anti-EZH2 (1:500; Abcam), rabbit anti-H3K27TM (1:500; Epigentek), rabbit anti-total histone H3 (1;1,000; cell signaling) primary antibodies, or a monoclonal rabbit anti-β-actin (1:2,000; Millipore) primary antibody as a loading control.

    Techniques: Expressing

    Pre-emptive DZNep or GSK126 treatment significantly attenuates the increased levels of EZH2 and H3K27TM in the spinal dorsal horn induced by nerve injury (A): Data were obtained from the spinal dorsal horn of animals treated with daily intrathecal injection of either 20 nM DZNep (in 10 μL) or vehicle (10 μL) for 9 days. (B): Data were obtained from the spinal dorsal horn of animals treated with daily intrathecal injection of either 5 nM GSK126 (in 10 μL) or vehicle (10 μL) for 9 days. Bar graphs show the mean (+ S.E.) relative density ratio of EZH2 over β-actin and H3K27TM over total-histone H3. Western blot samples of each molecular protein in each group are displayed. * p

    Journal: Neuroscience

    Article Title: EZH2 regulates spinal neuroinflammation in rats with neuropathic pain

    doi: 10.1016/j.neuroscience.2017.02.041

    Figure Lengend Snippet: Pre-emptive DZNep or GSK126 treatment significantly attenuates the increased levels of EZH2 and H3K27TM in the spinal dorsal horn induced by nerve injury (A): Data were obtained from the spinal dorsal horn of animals treated with daily intrathecal injection of either 20 nM DZNep (in 10 μL) or vehicle (10 μL) for 9 days. (B): Data were obtained from the spinal dorsal horn of animals treated with daily intrathecal injection of either 5 nM GSK126 (in 10 μL) or vehicle (10 μL) for 9 days. Bar graphs show the mean (+ S.E.) relative density ratio of EZH2 over β-actin and H3K27TM over total-histone H3. Western blot samples of each molecular protein in each group are displayed. * p

    Article Snippet: The membranes were blocked with 5% milk or 5% BSA in TBST, and then incubated respectively overnight at 4 °C with polyclonal rabbit anti-GFAP (1:1,000, cell signaling), polyclonal rabbit anti-Iba1 (1:1,000; Wako), rabbit anti-TNF-α (1:500; Millipore), rabbit anti-IL-1β (1:500; Millipore), rabbit anti-MCP-1 (1:500; Abcam), rabbit anti-EZH2 (1:500; Abcam), rabbit anti-H3K27TM (1:500; Epigentek), rabbit anti-total histone H3 (1;1,000; cell signaling) primary antibodies, or a monoclonal rabbit anti-β-actin (1:2,000; Millipore) primary antibody as a loading control.

    Techniques: Injection, Western Blot

    Nerve injury increases EZH2 protein expression and H3K27TM levels in spinal dorsal horn Bar graphs show the mean (+S.E.) of relative density ratio of EZH2 over β-actin and H3K27TM over total-histone H3 in the spinal dorsal horn of rats receiving either sham operation or pSNL on day 3 (A) and day 10 (B) after surgery. Western blot samples of each molecular protein in each group are displayed. * p

    Journal: Neuroscience

    Article Title: EZH2 regulates spinal neuroinflammation in rats with neuropathic pain

    doi: 10.1016/j.neuroscience.2017.02.041

    Figure Lengend Snippet: Nerve injury increases EZH2 protein expression and H3K27TM levels in spinal dorsal horn Bar graphs show the mean (+S.E.) of relative density ratio of EZH2 over β-actin and H3K27TM over total-histone H3 in the spinal dorsal horn of rats receiving either sham operation or pSNL on day 3 (A) and day 10 (B) after surgery. Western blot samples of each molecular protein in each group are displayed. * p

    Article Snippet: The membranes were blocked with 5% milk or 5% BSA in TBST, and then incubated respectively overnight at 4 °C with polyclonal rabbit anti-GFAP (1:1,000, cell signaling), polyclonal rabbit anti-Iba1 (1:1,000; Wako), rabbit anti-TNF-α (1:500; Millipore), rabbit anti-IL-1β (1:500; Millipore), rabbit anti-MCP-1 (1:500; Abcam), rabbit anti-EZH2 (1:500; Abcam), rabbit anti-H3K27TM (1:500; Epigentek), rabbit anti-total histone H3 (1;1,000; cell signaling) primary antibodies, or a monoclonal rabbit anti-β-actin (1:2,000; Millipore) primary antibody as a loading control.

    Techniques: Expressing, Western Blot