anti β actin  (Millipore)


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    Structured Review

    Millipore anti β actin
    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with <t>β-actin.</t> (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P
    Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti β actin - by Bioz Stars, 2020-09
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    Images

    1) Product Images from "MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion"

    Article Title: MUC1 is a downstream target of STAT3 and regulates lung cancer cell survival and invasion

    Journal: International journal of oncology

    doi:

    MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P
    Figure Legend Snippet: MUC1 is widely overexpressed in NSCLC cells, correlating with STAT3 activation. (A) Protein expression of MUC1-C and total and Tyr705 phosphorylated STAT3 levels were determined in 14 human NSCLC cell lines by Western blot analyses. Equal loading and transfer were shown by repeat probing with β-actin. (B) mRNA levels of MUC1 in a subset of cells were evaluated with real-time RT-PCR. MUC1 mRNA expression levels in each cell line were normalized to GAPDH mRNA, with expression in A549 cells set to an arbitrary value of 1. * P

    Techniques Used: Activation Assay, Expressing, Western Blot, Quantitative RT-PCR

    Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.
    Figure Legend Snippet: Effects of MUC1 knockdown or overexpression on multiple cell signals in NSCLC cells. (A) H358, HCC827, and H441 cells were exposed to siRNA for 72 h, and (B) A549 cells were transfected with MUC1-C plasmid for 48 h followed by measurement of phosphorylated tyrosine STAT3, Akt, Src, FAK, and apoptotic-related proteins by Western blot analysis. Equal loading and transfer were shown by repeat probing with β-actin.

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Western Blot

    2) Product Images from "Dual farnesyl and geranylgeranyl transferase inhibitor thwarts mutant KRAS-driven patient-derived pancreatic tumors"

    Article Title: Dual farnesyl and geranylgeranyl transferase inhibitor thwarts mutant KRAS-driven patient-derived pancreatic tumors

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    doi: 10.1158/1078-0432.CCR-18-3399

    FGTI-2734 inhibits oncogenic pathways mediated by AKT, mTOR, and cMYC, upregulates the tumor suppressor p53, and activates CASPASE 3 in patient-derived xenografts in vivo. Tumor tissue lysates from 6 vehicle-treated (mouse 1–6) and 7 FGTI-2734-treated (100mg/kg body weight; mouse 7–13) mice from patient 2 PDX were processed for Western blot analysis with indicated antibodies. A , Western blot results; superscripts a-e shown by antibodies correspond to loading controls containing vinculin or β-ACTIN. B and C , Quantification of Western blot results; letters a-f indicate statistical significance ( P
    Figure Legend Snippet: FGTI-2734 inhibits oncogenic pathways mediated by AKT, mTOR, and cMYC, upregulates the tumor suppressor p53, and activates CASPASE 3 in patient-derived xenografts in vivo. Tumor tissue lysates from 6 vehicle-treated (mouse 1–6) and 7 FGTI-2734-treated (100mg/kg body weight; mouse 7–13) mice from patient 2 PDX were processed for Western blot analysis with indicated antibodies. A , Western blot results; superscripts a-e shown by antibodies correspond to loading controls containing vinculin or β-ACTIN. B and C , Quantification of Western blot results; letters a-f indicate statistical significance ( P

    Techniques Used: Derivative Assay, In Vivo, Mouse Assay, Western Blot

    3) Product Images from "Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer"

    Article Title: Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-13-62

    TXNIP is highly expressed in DTC cell lines and low or undetectable in ATC cell lines, and glucose uptake is inversely proportional to TXNIP expression levels. (A) HTh74 cells were transduced with lentivirus expressing a PPARγ-specific shRNA or scrambled control, and stable pools were generated under antibiotic selection. Western blot analysis of nuclear lysates (top immunoblot) reveals PPARγ expression levels in the transduced cells. Using Western blot analysis of whole cell lysates, TXNIP and β-actin (loading control) were detected using specific antibodies. Nonspecific band is indicated by “ns”. (B) Western blot analysis for TXNIP, Trx-1, and β-actin (loading control) were performed on whole cell lysates prepared from a panel of DTC and ATC cell lines grown under standard conditions. (C) Glucose uptake assays were performed. Each condition was performed in triplicate per experiment, and each experiment was performed at least three times. Nonspecific glucose uptake as determined by parallel treatment of a subset with cytochalasin B was subtracted from measurements. Data from all experiments were combined, and glucose uptake from each cell line was normalized to levels of HTh74 (average set at 1). Normalized averages are plotted, and error bars indicate SEM. ***p
    Figure Legend Snippet: TXNIP is highly expressed in DTC cell lines and low or undetectable in ATC cell lines, and glucose uptake is inversely proportional to TXNIP expression levels. (A) HTh74 cells were transduced with lentivirus expressing a PPARγ-specific shRNA or scrambled control, and stable pools were generated under antibiotic selection. Western blot analysis of nuclear lysates (top immunoblot) reveals PPARγ expression levels in the transduced cells. Using Western blot analysis of whole cell lysates, TXNIP and β-actin (loading control) were detected using specific antibodies. Nonspecific band is indicated by “ns”. (B) Western blot analysis for TXNIP, Trx-1, and β-actin (loading control) were performed on whole cell lysates prepared from a panel of DTC and ATC cell lines grown under standard conditions. (C) Glucose uptake assays were performed. Each condition was performed in triplicate per experiment, and each experiment was performed at least three times. Nonspecific glucose uptake as determined by parallel treatment of a subset with cytochalasin B was subtracted from measurements. Data from all experiments were combined, and glucose uptake from each cell line was normalized to levels of HTh74 (average set at 1). Normalized averages are plotted, and error bars indicate SEM. ***p

    Techniques Used: Expressing, Transduction, shRNA, Generated, Selection, Western Blot

    TXNIP overexpression in ATC HTh74 and T238 cells attenuates glucose uptake. HTh74 and T238 cells were transduced with retrovirus encoding human TXNIP or vector control as well as a selectable antibiotic resistance marker, and stable pools were generated under antibiotic selection. Western blot analysis of whole cell lysates with TXNIP- and β-actin-specific antibodies is shown for HTh74 (A) and T238 (B) . Glucose uptake assays were performed as described in Figure 1 using the HTh74 stable cell lines (C) and T238 stable cell lines (D) . Data from all experiments were combined, and glucose uptake from each cell line was normalized to vector control levels (average set at 1). **p = 0.001, ***p
    Figure Legend Snippet: TXNIP overexpression in ATC HTh74 and T238 cells attenuates glucose uptake. HTh74 and T238 cells were transduced with retrovirus encoding human TXNIP or vector control as well as a selectable antibiotic resistance marker, and stable pools were generated under antibiotic selection. Western blot analysis of whole cell lysates with TXNIP- and β-actin-specific antibodies is shown for HTh74 (A) and T238 (B) . Glucose uptake assays were performed as described in Figure 1 using the HTh74 stable cell lines (C) and T238 stable cell lines (D) . Data from all experiments were combined, and glucose uptake from each cell line was normalized to vector control levels (average set at 1). **p = 0.001, ***p

    Techniques Used: Over Expression, Transduction, Plasmid Preparation, Marker, Generated, Selection, Western Blot, Stable Transfection

    4) Product Images from "Interrelation of inflammation and APP in sIBM: IL-1? induces accumulation of ?-amyloid in skeletal muscle"

    Article Title: Interrelation of inflammation and APP in sIBM: IL-1? induces accumulation of ?-amyloid in skeletal muscle

    Journal: Brain

    doi: 10.1093/brain/awn053

    Overexpression of APP and accumulation of β-amyloid in human primary skeletal myotube cultures exposed to IL-1β (10 ng/ml) and/or IFN-γ (300 U/ml) for 24–72 h. ( A ) A 24 h exposure to IL-1β, alone or in combination with IFN-γ, induced enhanced staining for APP (Alexa-594, red). Grey-scale analysis of the same experiment demonstrates a significant increase of the staining intensity upon IL-1β, and even more in combination with IFN-γ. ( B ) Immunoblot analysis of APP demonstrates an increased level of protein expression upon 24–72 h of exposure to IL-1β in combination with IFN-γ. Protein loading in each lane is confirmed by detection of β-actin. ( C ) Staining for β-amyloid reveals an enhanced intracellular accumulation upon 48 h of exposure to IL-1β, particularly in combination with IFN-γ (Alexa-488, green). Grey-scale analysis of the same experiment demonstrates a significant increase of the staining intensity upon IL-1β, and even more in combination with IFN-γ. ( D ) After 48 h of exposure to IL-1β and IFN-γ, intracellular aggregation of β-amyloid is evidenced by thioflavin-S, which is statistically significant as revealed by grey-scale analysis of the same experiment. Photos taken by a CCD-camera using a conventional fluorescent microscope with a 10× ( A and C ) or 20× ( D ) objective. All photomicrographs for the analyses in this Figure have been acquired with the same settings of camera and microscope; all data are representative of at least three experiments with similar results. Scale bars represent 100 µm in A, C and 60 µm in D .
    Figure Legend Snippet: Overexpression of APP and accumulation of β-amyloid in human primary skeletal myotube cultures exposed to IL-1β (10 ng/ml) and/or IFN-γ (300 U/ml) for 24–72 h. ( A ) A 24 h exposure to IL-1β, alone or in combination with IFN-γ, induced enhanced staining for APP (Alexa-594, red). Grey-scale analysis of the same experiment demonstrates a significant increase of the staining intensity upon IL-1β, and even more in combination with IFN-γ. ( B ) Immunoblot analysis of APP demonstrates an increased level of protein expression upon 24–72 h of exposure to IL-1β in combination with IFN-γ. Protein loading in each lane is confirmed by detection of β-actin. ( C ) Staining for β-amyloid reveals an enhanced intracellular accumulation upon 48 h of exposure to IL-1β, particularly in combination with IFN-γ (Alexa-488, green). Grey-scale analysis of the same experiment demonstrates a significant increase of the staining intensity upon IL-1β, and even more in combination with IFN-γ. ( D ) After 48 h of exposure to IL-1β and IFN-γ, intracellular aggregation of β-amyloid is evidenced by thioflavin-S, which is statistically significant as revealed by grey-scale analysis of the same experiment. Photos taken by a CCD-camera using a conventional fluorescent microscope with a 10× ( A and C ) or 20× ( D ) objective. All photomicrographs for the analyses in this Figure have been acquired with the same settings of camera and microscope; all data are representative of at least three experiments with similar results. Scale bars represent 100 µm in A, C and 60 µm in D .

    Techniques Used: Over Expression, Staining, Expressing, Microscopy

    5) Product Images from "Novel 5? Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease"

    Article Title: Novel 5? Untranslated Region Directed Blockers of Iron-Regulatory Protein-1 Dependent Amyloid Precursor Protein Translation: Implications for Down Syndrome and Alzheimer's Disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065978

    The effect of JTR-009 to reduce the steady state levels of APP in SH-SY5Y cells with a high degree of selectivity in the absence of changes to the levels of β-actin and α-synuclein ( SNCA ). Panels A and B : Dose-responsive (0, 10 µM, 20 µM, 30 µM) treatment of SH-SY5Y cells for 48 h to measure the capacity of JTR-009 and PFT-α to limit APP expression relative to β-actin and SNCA levels. The representative western blot experiment in Panel A contributed to densitometry for the histogram shown in Panel B (N = 3). Right Panel: Chemical structure of JTR-009, 4-(5-methyl-1H-benzimidazol-2yl) aniline, compared to the anti-apoptotic stroke agent PFTα, (275 Da), a tricyclic benzothiazole. Panel C : Dose-responsive measurement of total amyloid Aβ levels in response to the APP 5′UTR inhibitors JTR-005 and JTR-009, measured by benchmarked ELISA in conditioned medium of 72-hour treated SH-SY5Y cells. Shown are the mean values for the reduction of levels of Aβ ± SEM (N = 4) after treatment of the cells with JTR-009 and JTR-005 at 0.01 µM (* = p
    Figure Legend Snippet: The effect of JTR-009 to reduce the steady state levels of APP in SH-SY5Y cells with a high degree of selectivity in the absence of changes to the levels of β-actin and α-synuclein ( SNCA ). Panels A and B : Dose-responsive (0, 10 µM, 20 µM, 30 µM) treatment of SH-SY5Y cells for 48 h to measure the capacity of JTR-009 and PFT-α to limit APP expression relative to β-actin and SNCA levels. The representative western blot experiment in Panel A contributed to densitometry for the histogram shown in Panel B (N = 3). Right Panel: Chemical structure of JTR-009, 4-(5-methyl-1H-benzimidazol-2yl) aniline, compared to the anti-apoptotic stroke agent PFTα, (275 Da), a tricyclic benzothiazole. Panel C : Dose-responsive measurement of total amyloid Aβ levels in response to the APP 5′UTR inhibitors JTR-005 and JTR-009, measured by benchmarked ELISA in conditioned medium of 72-hour treated SH-SY5Y cells. Shown are the mean values for the reduction of levels of Aβ ± SEM (N = 4) after treatment of the cells with JTR-009 and JTR-005 at 0.01 µM (* = p

    Techniques Used: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

    Evaluation of the potency and selectivity of APP blocker-9. Panel A : Dose responsive measurement of the capacity of JTR-009 to limit APP 5′UTR-luciferase expression relative to posiphen, a known APP translation blocker (JTR-009: IC 50 = 0.1 µM; posiphen: IC 50 = 5 µM, N = 4). Panel B : Dose-responsive reduction APP levels in SH-SY5Y cells treated 48 hours at 0.1 µM, 0.5 µM and 1 µM JTR-009. Western blot for APP levels using N- terminal 22C11 antibody (standardization with β-actin as loading control). Bottom Panel: Histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells. Panel C : Lysates from the experiment in Panel B was analyzed by Western blotting using APP the C-terminal specific (A8717) antibody and β-actin antibody. Bottom Panel: histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells from autoradiographic film subjected to densitometry (N = 3). Panel D : Dose-responsive capacity of JTR-009 to limit APP expression in primary E-18 mouse neurons (1 nM). The relative α-synuclein ( SNCA ) expression was calculated. Shown, the combined data was graphed into a histogram where mean values from separate assays were calculated from densitometry of Western blots (N = 5). Panel E : Real-time qPCR measurement of the dose-responsive measurement of the levels of APP mRNA in SH-SY5Y cells treated with escalating concentrations of JTR-009 for 48 hours. Panel F : Equivalent real-time qRT-PCR analysis to measure APP mRNA and TfR mRNA levels in SH-SY5Y cells after 48 h treatment with 25 µM desferrioxamine (DFO) (Positive control for qRT-PCR analysis shown in Panel E).
    Figure Legend Snippet: Evaluation of the potency and selectivity of APP blocker-9. Panel A : Dose responsive measurement of the capacity of JTR-009 to limit APP 5′UTR-luciferase expression relative to posiphen, a known APP translation blocker (JTR-009: IC 50 = 0.1 µM; posiphen: IC 50 = 5 µM, N = 4). Panel B : Dose-responsive reduction APP levels in SH-SY5Y cells treated 48 hours at 0.1 µM, 0.5 µM and 1 µM JTR-009. Western blot for APP levels using N- terminal 22C11 antibody (standardization with β-actin as loading control). Bottom Panel: Histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells. Panel C : Lysates from the experiment in Panel B was analyzed by Western blotting using APP the C-terminal specific (A8717) antibody and β-actin antibody. Bottom Panel: histogram quantitation of the relative expression of APP/β-actin in SH-SY5Y cells from autoradiographic film subjected to densitometry (N = 3). Panel D : Dose-responsive capacity of JTR-009 to limit APP expression in primary E-18 mouse neurons (1 nM). The relative α-synuclein ( SNCA ) expression was calculated. Shown, the combined data was graphed into a histogram where mean values from separate assays were calculated from densitometry of Western blots (N = 5). Panel E : Real-time qPCR measurement of the dose-responsive measurement of the levels of APP mRNA in SH-SY5Y cells treated with escalating concentrations of JTR-009 for 48 hours. Panel F : Equivalent real-time qRT-PCR analysis to measure APP mRNA and TfR mRNA levels in SH-SY5Y cells after 48 h treatment with 25 µM desferrioxamine (DFO) (Positive control for qRT-PCR analysis shown in Panel E).

    Techniques Used: Luciferase, Expressing, Western Blot, Quantitation Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Positive Control

    6) Product Images from "Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy"

    Article Title: Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy

    Journal: Nature Communications

    doi: 10.1038/ncomms16078

    HTS of a small molecule pathway inhibitor library identifies CDK9 inhibitors that enhance the proapoptotic effect of ABT-737. ( a ) A schematic of HTS of the pathway inhibitor library to identify agents that cooperate with ABT-737 to kill H196 cells. H196 cells were used to screen the pathway inhibitor library at 2 μM±1 μM ABT-737. Compounds were screened in duplicate and cell viability was assessed by Alamar Blue assays at 72 h. ( b ) Scatter plot analysis of the average percentage growth inhibition of each compound screened in H196 cells. The compounds that induced ≤50% growth inhibition in the absence of ABT-737 while ≥50% inhibition in the presence of ABT-737 were identified as positive hits (red dots). ( c ) A summary of hit compounds identified by the HTS of the pathway inhibitor library. ( d ) H196 cells, transfected with scrambled siRNA (si SCR ) or siRNA against CDK9 , were treated with 1 μM ABT-263 and subjected to cell death or immunoblot analysis. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( e ) H196 cells, treated with the indicated agents for 3 h, were assessed by immunoblot analysis. ( f ) H196 cells were treated with the indicated agents±1 μM ABT-737 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( g ) The mRNA levels of BCL-2 family in H196 cells treated with 10 nM dinaciclib for 3 h were assessed by qRT-PCR. Data were normalized against β-Actin (mean±s.d., n =2 independent experiments). ( h ) H196 cells, untreated or treated with the indicated CDK9 inhibitors for 6 h, were assessed by immunoblot analysis. ( i ) The indicated SCLC cell lines were treated with vehicle or dinaciclib (10 nM for H196, H82, and H446, or 20 nM for SW1271 and DMS114) in the absence or presence of 1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ** P
    Figure Legend Snippet: HTS of a small molecule pathway inhibitor library identifies CDK9 inhibitors that enhance the proapoptotic effect of ABT-737. ( a ) A schematic of HTS of the pathway inhibitor library to identify agents that cooperate with ABT-737 to kill H196 cells. H196 cells were used to screen the pathway inhibitor library at 2 μM±1 μM ABT-737. Compounds were screened in duplicate and cell viability was assessed by Alamar Blue assays at 72 h. ( b ) Scatter plot analysis of the average percentage growth inhibition of each compound screened in H196 cells. The compounds that induced ≤50% growth inhibition in the absence of ABT-737 while ≥50% inhibition in the presence of ABT-737 were identified as positive hits (red dots). ( c ) A summary of hit compounds identified by the HTS of the pathway inhibitor library. ( d ) H196 cells, transfected with scrambled siRNA (si SCR ) or siRNA against CDK9 , were treated with 1 μM ABT-263 and subjected to cell death or immunoblot analysis. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( e ) H196 cells, treated with the indicated agents for 3 h, were assessed by immunoblot analysis. ( f ) H196 cells were treated with the indicated agents±1 μM ABT-737 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( g ) The mRNA levels of BCL-2 family in H196 cells treated with 10 nM dinaciclib for 3 h were assessed by qRT-PCR. Data were normalized against β-Actin (mean±s.d., n =2 independent experiments). ( h ) H196 cells, untreated or treated with the indicated CDK9 inhibitors for 6 h, were assessed by immunoblot analysis. ( i ) The indicated SCLC cell lines were treated with vehicle or dinaciclib (10 nM for H196, H82, and H446, or 20 nM for SW1271 and DMS114) in the absence or presence of 1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ** P

    Techniques Used: Inhibition, Transfection, Staining, Quantitative RT-PCR

    Dinaciclib but not JQ1 synergizes with ABT-263 to kill SCLC cells. ( a ) The indicated SCLC cell lines were treated with vehicle, 1 μM JQ1, or dinaciclib (10 nM for H196 and H82, or 20 nM for SW1271 and DMS114) in the absence or presence of 1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( b ) A summary of EC50s of dinaciclib and JQ1 in SCLC cell lines. The indicated SCLC cell lines were untreated or treated with increasing concentrations of dinaciclib or JQ1. Cell viability was assessed by CellTiter-Glo assays at 72 h. ( c ) The expression of c-MYC in the indicated SCLC cell lines was assessed by an anti-c-Myc immunoblot. ( d ) The indicated SCLC cell lines were treated with vehicle, 1 μM JQ1, or 20 nM dinaciclib for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( e ) H82, H2171, and H446 cells, treated with vehicle, 20 nM dinaciclib, or 1 μM JQ1 for 3 h, were assessed by qRT-PCR. Data were normalized against β-actin (mean±s.d., n =2 independent experiments). H82, H2171, and H446 cells, treated with vehicle, dinaciclib, or JQ1 for 24 h, were assessed by immunoblot analysis using the indicated antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 11 .
    Figure Legend Snippet: Dinaciclib but not JQ1 synergizes with ABT-263 to kill SCLC cells. ( a ) The indicated SCLC cell lines were treated with vehicle, 1 μM JQ1, or dinaciclib (10 nM for H196 and H82, or 20 nM for SW1271 and DMS114) in the absence or presence of 1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( b ) A summary of EC50s of dinaciclib and JQ1 in SCLC cell lines. The indicated SCLC cell lines were untreated or treated with increasing concentrations of dinaciclib or JQ1. Cell viability was assessed by CellTiter-Glo assays at 72 h. ( c ) The expression of c-MYC in the indicated SCLC cell lines was assessed by an anti-c-Myc immunoblot. ( d ) The indicated SCLC cell lines were treated with vehicle, 1 μM JQ1, or 20 nM dinaciclib for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( e ) H82, H2171, and H446 cells, treated with vehicle, 20 nM dinaciclib, or 1 μM JQ1 for 3 h, were assessed by qRT-PCR. Data were normalized against β-actin (mean±s.d., n =2 independent experiments). H82, H2171, and H446 cells, treated with vehicle, dinaciclib, or JQ1 for 24 h, were assessed by immunoblot analysis using the indicated antibodies. Unprocessed original scans of blots are shown in Supplementary Fig. 11 .

    Techniques Used: Staining, Expressing, Quantitative RT-PCR

    HTS of FDA-approved anti-cancer agents identifies anthracyclines that enhance the proapoptotic effect of ABT-737. ( a ) DMS53 and H196 cells were treated with the indicated agents for 48 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( b ) The expression of BCL-2 family proteins in DMS53 and H196 cells was assessed by immunoblot analysis. ( c ) A schematic of HTS to identify FDA-approved anti-cancer agents that cooperate with ABT-737 to reduce the survival of H196 cells. H196 cells were treated with each compound at 12 two-fold serial dilution doses starting from 10±1 μM ABT-737. Cell viability was assessed by Alamar Blue assays at 72 h and EC50 was calculated. ( d ) A summary of EC50s of anti-cancer agents±ABT-737 in H196 cells. NA denotes ‘not applicable’, because the agent failed to achieve 100% growth inhibition at 10 μM. ( e ) H196 cells were treated with the indicated agents for 48 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( f ) H196 treated with the indicated agents for 6 h were assessed by immunoblot analysis. ( g ) The mRNA levels of BCL-2 family in H196 cells treated with 2 μM doxorubicin for 3 h were assessed by qRT–PCR. Data were normalized against β-Actin (mean±s.d., n =2 independent experiments). ( h ) H196 cells treated with doxorubicin for the indicated times were assessed by immunoblot analysis. ( i ) A summary of EC50s of ABT-263 in SCLC cell lines. The indicated SCLC cell lines were treated with increasing concentrations of ABT-263. Cell viability was assessed by CellTiter-Glo assays at 48 h. DMS53 and H2171 cells were treated with 1 μM ABT-263 for 24 h and cell death was quantified by annexin-V staining (mean±s.d., n =3). ( j ) The indicated SCLC cell lines were treated with 2 μM doxorubicin±1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). * P
    Figure Legend Snippet: HTS of FDA-approved anti-cancer agents identifies anthracyclines that enhance the proapoptotic effect of ABT-737. ( a ) DMS53 and H196 cells were treated with the indicated agents for 48 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( b ) The expression of BCL-2 family proteins in DMS53 and H196 cells was assessed by immunoblot analysis. ( c ) A schematic of HTS to identify FDA-approved anti-cancer agents that cooperate with ABT-737 to reduce the survival of H196 cells. H196 cells were treated with each compound at 12 two-fold serial dilution doses starting from 10±1 μM ABT-737. Cell viability was assessed by Alamar Blue assays at 72 h and EC50 was calculated. ( d ) A summary of EC50s of anti-cancer agents±ABT-737 in H196 cells. NA denotes ‘not applicable’, because the agent failed to achieve 100% growth inhibition at 10 μM. ( e ) H196 cells were treated with the indicated agents for 48 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). ( f ) H196 treated with the indicated agents for 6 h were assessed by immunoblot analysis. ( g ) The mRNA levels of BCL-2 family in H196 cells treated with 2 μM doxorubicin for 3 h were assessed by qRT–PCR. Data were normalized against β-Actin (mean±s.d., n =2 independent experiments). ( h ) H196 cells treated with doxorubicin for the indicated times were assessed by immunoblot analysis. ( i ) A summary of EC50s of ABT-263 in SCLC cell lines. The indicated SCLC cell lines were treated with increasing concentrations of ABT-263. Cell viability was assessed by CellTiter-Glo assays at 48 h. DMS53 and H2171 cells were treated with 1 μM ABT-263 for 24 h and cell death was quantified by annexin-V staining (mean±s.d., n =3). ( j ) The indicated SCLC cell lines were treated with 2 μM doxorubicin±1 μM ABT-263 for 24 h. Cell death was quantified by annexin-V staining (mean±s.d., n =3). * P

    Techniques Used: Staining, Expressing, Serial Dilution, Inhibition, Quantitative RT-PCR

    7) Product Images from "NSPc1 is a cell growth regulator that acts as a transcriptional repressor of p21Waf1/Cip1 via the RARE element"

    Article Title: NSPc1 is a cell growth regulator that acts as a transcriptional repressor of p21Waf1/Cip1 via the RARE element

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl834

    Effects of NSPc1 on HeLa cell cycle progression, cell proliferation and cell colony formation. ( a ) Establishment of the NSPc1 stably over-expressed and NSPc1 knocked-down HeLa cell pools. Up: changes in mRNA level of HeLa stably transfected cells pool confirmed by semi-quantitative RT–PCR. PCR products generated by primer sets for NSPc1 were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Down: changes in protein expression confirmed by Western blots (normalized by β-actin protein level). ( b and c ) Flow cytometric analysis of HeLa stable cell pools. ( d and e ) MTT assay of HeLa stable cell pools. ( f and g ) Colony formation assay. Culture dishes fixed and stained with 0.1% crystal violet at the end of the experiment. The numbers of total colonies and colonies greater than 2 mm in diameter were counted and the means and SDs of three independent experiments were shown. Abbreviations: V5, pcDNA3.1-V5 cell pool; NSPc1, pcDNA3.1-V5+NSPc1 cell pool; IMG800, IMG800 cell pool; RNAi1, NSPc1-RNAi1 cell pool; RNAi2, NSPc1-RNAi2 cell pool. Note: a representative experiment out of three performed is shown in (b–f).
    Figure Legend Snippet: Effects of NSPc1 on HeLa cell cycle progression, cell proliferation and cell colony formation. ( a ) Establishment of the NSPc1 stably over-expressed and NSPc1 knocked-down HeLa cell pools. Up: changes in mRNA level of HeLa stably transfected cells pool confirmed by semi-quantitative RT–PCR. PCR products generated by primer sets for NSPc1 were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH); Down: changes in protein expression confirmed by Western blots (normalized by β-actin protein level). ( b and c ) Flow cytometric analysis of HeLa stable cell pools. ( d and e ) MTT assay of HeLa stable cell pools. ( f and g ) Colony formation assay. Culture dishes fixed and stained with 0.1% crystal violet at the end of the experiment. The numbers of total colonies and colonies greater than 2 mm in diameter were counted and the means and SDs of three independent experiments were shown. Abbreviations: V5, pcDNA3.1-V5 cell pool; NSPc1, pcDNA3.1-V5+NSPc1 cell pool; IMG800, IMG800 cell pool; RNAi1, NSPc1-RNAi1 cell pool; RNAi2, NSPc1-RNAi2 cell pool. Note: a representative experiment out of three performed is shown in (b–f).

    Techniques Used: Stable Transfection, Transfection, Quantitative RT-PCR, Polymerase Chain Reaction, Generated, Expressing, Western Blot, Flow Cytometry, MTT Assay, Colony Assay, Staining

    NSPc1 specifically represses the expression of the CDK inhibitor p21. ( a ) Semi-quantitative RT–PCR results (normalized by GAPDH level) show the expression level of CDKIs in various HeLa stable cell pools. Bmi-1 was detected as a control of NSPc1-RNAi specificity. Similar results were observed in three independent experiments. ( b ) Western blot results confirmed that the expression levels of p21 protein in various stable cell pools were reversely correlated with NSPc1 (normalized by β-actin protein level).
    Figure Legend Snippet: NSPc1 specifically represses the expression of the CDK inhibitor p21. ( a ) Semi-quantitative RT–PCR results (normalized by GAPDH level) show the expression level of CDKIs in various HeLa stable cell pools. Bmi-1 was detected as a control of NSPc1-RNAi specificity. Similar results were observed in three independent experiments. ( b ) Western blot results confirmed that the expression levels of p21 protein in various stable cell pools were reversely correlated with NSPc1 (normalized by β-actin protein level).

    Techniques Used: Expressing, Quantitative RT-PCR, Stable Transfection, Western Blot

    8) Product Images from "Morphological characterization of NG2 glia and their association with neuroglial cells in the 3-nitropropionic acid–lesioned striatum of rat"

    Article Title: Morphological characterization of NG2 glia and their association with neuroglial cells in the 3-nitropropionic acid–lesioned striatum of rat

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24385-0

    Representative images showing the temporal profiles of NG2-positive cells in 3-NP-treated rat forebrains. ( a ) Low-magnification view of a coronal section from a saline-treated control. ( b ) Higher-magnification view of the boxed area shown in a. Note that weak NG2 expression was observed in small stellate cells with fine processes in control striatum. ( c – e ) Prominent NG2 immunoreactivity appeared at the lesion edge by day 1 ( c ), and appeared to move into the epicenter on days 2 ( d ) and 3 ( e ) post-lesion. The boxed areas in e and g are enlarged in g and h, respectively. Note that NG2-positive cells in the lesion periphery had irregular cell bodies with thick and short processes (arrows in h), while small stellate cells with long fine processes (arrowhead in h) were preferentially found in the epicenter. ( f ) Merged image of FJB staining and GFAP immunoreactivity using serial striatal sections obtained 3 days post-lesion. Note that the lesion core is distinguished by intense FJB staining and concomitant loss of GFAP-positive astrocytes. ( i – k ) Changes in NG2 immunoreactivity in striatal sections from 3-NP-treated rats 7 ( i ), 14 ( j ), and 28 days (k) post-lesion. Prominent NG2 immunoreactivity was localized within both the lesion edge and the epicenter. LV: lateral ventricle. ( l ) Representative western blot analysis results for NG2 protein expression in striatal extracts from sham controls and rats killed 7, 14, and 28 days post-lesion. Note that a band of about 300 kDa corresponding to NG2 protein was clearly observed in both the control and lesioned striatum. ( m ) Quantification of NG2 protein expression. Data were obtained using densitometry and were normalized using β-actin as the loading control. The results are expressed in relative optical density and represent means ± SEM. The intensity of NG2 protein reactivity significantly increased by 14 days after 3-NP injection and then decreased, although enhanced expression levels persisted until at least day 28. **P
    Figure Legend Snippet: Representative images showing the temporal profiles of NG2-positive cells in 3-NP-treated rat forebrains. ( a ) Low-magnification view of a coronal section from a saline-treated control. ( b ) Higher-magnification view of the boxed area shown in a. Note that weak NG2 expression was observed in small stellate cells with fine processes in control striatum. ( c – e ) Prominent NG2 immunoreactivity appeared at the lesion edge by day 1 ( c ), and appeared to move into the epicenter on days 2 ( d ) and 3 ( e ) post-lesion. The boxed areas in e and g are enlarged in g and h, respectively. Note that NG2-positive cells in the lesion periphery had irregular cell bodies with thick and short processes (arrows in h), while small stellate cells with long fine processes (arrowhead in h) were preferentially found in the epicenter. ( f ) Merged image of FJB staining and GFAP immunoreactivity using serial striatal sections obtained 3 days post-lesion. Note that the lesion core is distinguished by intense FJB staining and concomitant loss of GFAP-positive astrocytes. ( i – k ) Changes in NG2 immunoreactivity in striatal sections from 3-NP-treated rats 7 ( i ), 14 ( j ), and 28 days (k) post-lesion. Prominent NG2 immunoreactivity was localized within both the lesion edge and the epicenter. LV: lateral ventricle. ( l ) Representative western blot analysis results for NG2 protein expression in striatal extracts from sham controls and rats killed 7, 14, and 28 days post-lesion. Note that a band of about 300 kDa corresponding to NG2 protein was clearly observed in both the control and lesioned striatum. ( m ) Quantification of NG2 protein expression. Data were obtained using densitometry and were normalized using β-actin as the loading control. The results are expressed in relative optical density and represent means ± SEM. The intensity of NG2 protein reactivity significantly increased by 14 days after 3-NP injection and then decreased, although enhanced expression levels persisted until at least day 28. **P

    Techniques Used: Expressing, Staining, Western Blot, Injection

    9) Product Images from "Interplay between Epigenetics, Expression of Estrogen Receptor- α, HER2/ERBB2 and Sensitivity of Triple Negative Breast Cancer Cells to Hormonal Therapy"

    Article Title: Interplay between Epigenetics, Expression of Estrogen Receptor- α, HER2/ERBB2 and Sensitivity of Triple Negative Breast Cancer Cells to Hormonal Therapy

    Journal: Cancers

    doi: 10.3390/cancers11010013

    Effect of suberoylanilide hydroxamic acid (SAHA) and 5-aza-dc on the expression of epigenetic markers in breast cancer cells. Analysis of DNMT1, HDAC1, 2, 3, 4, 6, and p-HDAC 4/5/7 proteins upon treatment with IC50 concentrations of SAHA for 72 h or/and 5-aza-dc for 96 h in ( A ) MCF7, ( B ) SkBr3, ( C ) BT-549, and ( D ) MDA-MB-231. DNMT1 and HDAC1 were visualized on the same blot, HDAC2, HDAC6 and phospho- HDAC4,5 and 7 were visualized on another blot whereas HDAC3 and 4 were visualized on a third blot. Bar graphs showing the relative fold change of DNMT1 and HDACs after normalization to β-actin expression and DMSO treatment. The level of each protein was normalized to the corresponding β-actin from the same blot. Shown are the means ± SEM of at least three independent experiments. * p
    Figure Legend Snippet: Effect of suberoylanilide hydroxamic acid (SAHA) and 5-aza-dc on the expression of epigenetic markers in breast cancer cells. Analysis of DNMT1, HDAC1, 2, 3, 4, 6, and p-HDAC 4/5/7 proteins upon treatment with IC50 concentrations of SAHA for 72 h or/and 5-aza-dc for 96 h in ( A ) MCF7, ( B ) SkBr3, ( C ) BT-549, and ( D ) MDA-MB-231. DNMT1 and HDAC1 were visualized on the same blot, HDAC2, HDAC6 and phospho- HDAC4,5 and 7 were visualized on another blot whereas HDAC3 and 4 were visualized on a third blot. Bar graphs showing the relative fold change of DNMT1 and HDACs after normalization to β-actin expression and DMSO treatment. The level of each protein was normalized to the corresponding β-actin from the same blot. Shown are the means ± SEM of at least three independent experiments. * p

    Techniques Used: Expressing, Multiple Displacement Amplification

    Effect of SAHA, 5-aza-dc and their combination on the expression of ERα in breast cancer cells. ( A – D ) Upper panels: Western blot analysis of ERα expression in ( A ) MCF7, ( B ) SkBr3, ( C ) BT549 and ( D ) MDA-MB-231 cells treated with the IC50 concentrations of SAHA and/or 5-aza-dc. Lower panel: Bar graphs showing relative fold changes of ERα bands after quantification and normalization to β-actin expression and DMSO treatment. ( E , F ) Representative micrographs (at 100× magnification) of immunofluorescence staining of ERα (green) and 4′,6-diamidino-2-phenylindole (DAPI)(blue) in ( E ) MCF7 and ( F ) BT-549 cells treated with SAHA and/or 5-aza-dc. ( G , F ) Western blots of ERα expression in ( G ) BT-549 and ( H ) MDA-MB-231 cells treated with IC25, IC50, and double IC50 (dIC50) concentrations of SAHA or 5-aza-dc. Shown are the means ± SEM of at least three independent experiments. * p
    Figure Legend Snippet: Effect of SAHA, 5-aza-dc and their combination on the expression of ERα in breast cancer cells. ( A – D ) Upper panels: Western blot analysis of ERα expression in ( A ) MCF7, ( B ) SkBr3, ( C ) BT549 and ( D ) MDA-MB-231 cells treated with the IC50 concentrations of SAHA and/or 5-aza-dc. Lower panel: Bar graphs showing relative fold changes of ERα bands after quantification and normalization to β-actin expression and DMSO treatment. ( E , F ) Representative micrographs (at 100× magnification) of immunofluorescence staining of ERα (green) and 4′,6-diamidino-2-phenylindole (DAPI)(blue) in ( E ) MCF7 and ( F ) BT-549 cells treated with SAHA and/or 5-aza-dc. ( G , F ) Western blots of ERα expression in ( G ) BT-549 and ( H ) MDA-MB-231 cells treated with IC25, IC50, and double IC50 (dIC50) concentrations of SAHA or 5-aza-dc. Shown are the means ± SEM of at least three independent experiments. * p

    Techniques Used: Expressing, Western Blot, Multiple Displacement Amplification, Immunofluorescence, Staining

    10) Product Images from "A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies"

    Article Title: A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies

    Journal: Reproductive biology and endocrinology : RB & E

    doi: 10.1186/1477-7827-3-25

    The production of mature LHCGR isoforms in chorinic villi from Down's syndrome pregnancies are significantly reduced compared to that of controls. The CVS samples were Tri-zol extracted to recover mRNA as well as proteins. Approximately 10 μg of total protein was loaded in each lane. The proteins extracted from DS (T21) CVS (a) and control CVS (b) pregnancies were resolved in 8% polyacrylamide-SDS gels, Western blotted and immunoreacted with anti-human LHCGR (LHR-29) monoclonal antibody. Blots were stripped prior to immunostaining with anti-β-Actin monoclonal antibody. The data shown in a) and b) were from the same experiment except that the control and DS proteins were separated in two gels at the same time. In order to compare the band intensity in different experiments, two known CVS samples in duplicate were incorporated in each experiment. The density of the 80K LHCGR and 42K β-actin bands served as references for quantitative analysis of the experimental samples. The relative migration of the isoforms is indicated by an arrow. The M r 80K protein band (LHCGR p80), indicated by * in a) and b), well separated from the neighboring variants were scanned and c) the relative densities of the LHCGR p80 with respect to β-Actin in normal and trisomic pregnancies, n = total number of experiments carried out on protein samples in each condition. ** P
    Figure Legend Snippet: The production of mature LHCGR isoforms in chorinic villi from Down's syndrome pregnancies are significantly reduced compared to that of controls. The CVS samples were Tri-zol extracted to recover mRNA as well as proteins. Approximately 10 μg of total protein was loaded in each lane. The proteins extracted from DS (T21) CVS (a) and control CVS (b) pregnancies were resolved in 8% polyacrylamide-SDS gels, Western blotted and immunoreacted with anti-human LHCGR (LHR-29) monoclonal antibody. Blots were stripped prior to immunostaining with anti-β-Actin monoclonal antibody. The data shown in a) and b) were from the same experiment except that the control and DS proteins were separated in two gels at the same time. In order to compare the band intensity in different experiments, two known CVS samples in duplicate were incorporated in each experiment. The density of the 80K LHCGR and 42K β-actin bands served as references for quantitative analysis of the experimental samples. The relative migration of the isoforms is indicated by an arrow. The M r 80K protein band (LHCGR p80), indicated by * in a) and b), well separated from the neighboring variants were scanned and c) the relative densities of the LHCGR p80 with respect to β-Actin in normal and trisomic pregnancies, n = total number of experiments carried out on protein samples in each condition. ** P

    Techniques Used: Western Blot, Immunostaining, Migration

    11) Product Images from "Peroxiredoxin 1 expression in active ulcerative colitis mucosa identified by proteome analysis and involvement of thioredoxin based on immunohistochemistry"

    Article Title: Peroxiredoxin 1 expression in active ulcerative colitis mucosa identified by proteome analysis and involvement of thioredoxin based on immunohistochemistry

    Journal: Oncology Letters

    doi: 10.3892/ol.2017.7549

    Western blot analysis of PRDX1 expression in protein extracts of sigmoid colon biopsy specimens (normal mucosa, inactive UC, and active UC) and colon cancer cell lysates. Two active UC samples showed higher PRDX1 expression than that in normal mucosa and inactive UC. Colon cancer cell lysates (UCCA-24: UC-associated colon cancer cells, KE43P: Sporadic colon cancer cells) also expressed PRDX1. β-actin expression was used as a control.
    Figure Legend Snippet: Western blot analysis of PRDX1 expression in protein extracts of sigmoid colon biopsy specimens (normal mucosa, inactive UC, and active UC) and colon cancer cell lysates. Two active UC samples showed higher PRDX1 expression than that in normal mucosa and inactive UC. Colon cancer cell lysates (UCCA-24: UC-associated colon cancer cells, KE43P: Sporadic colon cancer cells) also expressed PRDX1. β-actin expression was used as a control.

    Techniques Used: Western Blot, Expressing

    12) Product Images from "Degrons at the C Terminus of the Pathogenic but Not the Nonpathogenic Hantavirus G1 Tail Direct Proteasomal Degradation ▿"

    Article Title: Degrons at the C Terminus of the Pathogenic but Not the Nonpathogenic Hantavirus G1 Tail Direct Proteasomal Degradation ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02279-06

    The C-terminal 30 residues of NY-1V G1 tails direct proteasomal degradation. Deletion mutants lacking 5, 14, 30, or 51 residues from the NY-1V G1 tail C terminus were expressed in COS7 cells. Protein expression from MG132-treated (+) and untreated (−) cells was analyzed by Western blotting (WB) using an anti-GAL4 antibody. Blots were reprobed for β-actin to demonstrate comparable sample loading.
    Figure Legend Snippet: The C-terminal 30 residues of NY-1V G1 tails direct proteasomal degradation. Deletion mutants lacking 5, 14, 30, or 51 residues from the NY-1V G1 tail C terminus were expressed in COS7 cells. Protein expression from MG132-treated (+) and untreated (−) cells was analyzed by Western blotting (WB) using an anti-GAL4 antibody. Blots were reprobed for β-actin to demonstrate comparable sample loading.

    Techniques Used: Expressing, Western Blot

    G1 cytoplasmic tails of pathogenic HPS- and HFRS-causing hantaviruses are proteasomally degraded, while the G1 tail of nonpathogenic hantavirus is stable. One microgram of plasmid DNA expressing GAL4-tagged G1 tails of NY-1V, ANDV, HTNV, or PHV was transiently transfected into COS7 cells, and 42 h posttransfection, cells were either treated with 50 μM MG132 for 6 h (+) or left untreated (−). Lysate containing 10 μg of total protein was analyzed on 12% SDS-PAGE gels by immunoblotting with an anti-GAL4 antibody. β-Actin levels were evaluated to demonstrate comparable sample loading.
    Figure Legend Snippet: G1 cytoplasmic tails of pathogenic HPS- and HFRS-causing hantaviruses are proteasomally degraded, while the G1 tail of nonpathogenic hantavirus is stable. One microgram of plasmid DNA expressing GAL4-tagged G1 tails of NY-1V, ANDV, HTNV, or PHV was transiently transfected into COS7 cells, and 42 h posttransfection, cells were either treated with 50 μM MG132 for 6 h (+) or left untreated (−). Lysate containing 10 μg of total protein was analyzed on 12% SDS-PAGE gels by immunoblotting with an anti-GAL4 antibody. β-Actin levels were evaluated to demonstrate comparable sample loading.

    Techniques Used: Plasmid Preparation, Expressing, Transfection, SDS Page

    The degradation signal in the C-terminal 42 residues of the NY-1V G1 tail is independent of lysines at positions 615 and 628. COS7 cells were transfected with 1 μg of plasmid DNA expressing C42 lysine mutants, and 6 h prior to lysis, 50 μM MG132 was added to selected wells (+). Ten micrograms of total protein was analyzed on a 12% SDS-PAGE gel by anti-GAL4 immunoblotting (WB). Blots were reprobed for β-actin to demonstrate comparable sample loading.
    Figure Legend Snippet: The degradation signal in the C-terminal 42 residues of the NY-1V G1 tail is independent of lysines at positions 615 and 628. COS7 cells were transfected with 1 μg of plasmid DNA expressing C42 lysine mutants, and 6 h prior to lysis, 50 μM MG132 was added to selected wells (+). Ten micrograms of total protein was analyzed on a 12% SDS-PAGE gel by anti-GAL4 immunoblotting (WB). Blots were reprobed for β-actin to demonstrate comparable sample loading.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Lysis, SDS Page, Western Blot

    Stability-determining amino acids in pathogenic and nonpathogenic G1 tails. (A) Equivalent amounts of the NY-1V and PHV G1 tail mutants indicated were transiently expressed in COS7 cells in the presence (+) or absence (−) of MG132. Protein expression levels were determined by immunoblotting (WB) using anti-GAL4 antibodies. UT, untransfected COS7 lysate. Blots were reprobed for β-actin as an internal loading control. (B) Replacement of residues C1 to C7 and C4 to C7 in the NY-1V G1 tail with PHV residues enhances NY-1V G1 stability. (C) Mutations at residues C1 to C4 alter the stable PHV G1 tail into a proteasomally degraded chimera. Asterisk indicates a nonspecific cellular band.
    Figure Legend Snippet: Stability-determining amino acids in pathogenic and nonpathogenic G1 tails. (A) Equivalent amounts of the NY-1V and PHV G1 tail mutants indicated were transiently expressed in COS7 cells in the presence (+) or absence (−) of MG132. Protein expression levels were determined by immunoblotting (WB) using anti-GAL4 antibodies. UT, untransfected COS7 lysate. Blots were reprobed for β-actin as an internal loading control. (B) Replacement of residues C1 to C7 and C4 to C7 in the NY-1V G1 tail with PHV residues enhances NY-1V G1 stability. (C) Mutations at residues C1 to C4 alter the stable PHV G1 tail into a proteasomally degraded chimera. Asterisk indicates a nonspecific cellular band.

    Techniques Used: Expressing, Western Blot

    13) Product Images from "Establishment and Characterization of Rat Portal Myofibroblast Cell Lines"

    Article Title: Establishment and Characterization of Rat Portal Myofibroblast Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121161

    Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunofluorescence. Immortalized RGF cells were fixed, stained with antibodies to proteins specifically expressed in PF-derived myofibroblasts, and counterstained with DAPI nuclear labeling dye. RGF cells express myofibroblast-specific αSMA and Cd73 proteins, PF-specific elastin and Ntpdase2 proteins, β-actin protein and SV40 antigen. Immortalized RGF-N2 cells were fixed, stained with the same antibodies, and counterstained with DAPI dye, as described above for RGF cells. Like RGF cells, RGF-N2 cells express αSMA, Cd73, Elastin, Ntpdase2, β-actin, and SV40 antigen. 400X magnification .
    Figure Legend Snippet: Phenotypic characterization of immortalized rat portal fibroblastic RGF and RGF-N2 cell lines by immunofluorescence. Immortalized RGF cells were fixed, stained with antibodies to proteins specifically expressed in PF-derived myofibroblasts, and counterstained with DAPI nuclear labeling dye. RGF cells express myofibroblast-specific αSMA and Cd73 proteins, PF-specific elastin and Ntpdase2 proteins, β-actin protein and SV40 antigen. Immortalized RGF-N2 cells were fixed, stained with the same antibodies, and counterstained with DAPI dye, as described above for RGF cells. Like RGF cells, RGF-N2 cells express αSMA, Cd73, Elastin, Ntpdase2, β-actin, and SV40 antigen. 400X magnification .

    Techniques Used: Immunofluorescence, Staining, Derivative Assay, Labeling

    14) Product Images from "Novel Chimeric Protein Vaccines Against Clostridium difficile Infection"

    Article Title: Novel Chimeric Protein Vaccines Against Clostridium difficile Infection

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.02440

    Glucosyltransferase activity of TcdB is blocked by anti-Tcd169 or anti-Tcd169FI serum. (A) CT26 cells were lysed, and the cytosolic fraction was exposed to TcdB (10 ng/ml) with or without serum for 1 h followed by Western Blot analysis using a monoclonal antibody that only recognizes non-glucosylated Rac1. β-actin was used as an equal loading control. (B) Quantitation of Rac1 levels in (A) ( ** p
    Figure Legend Snippet: Glucosyltransferase activity of TcdB is blocked by anti-Tcd169 or anti-Tcd169FI serum. (A) CT26 cells were lysed, and the cytosolic fraction was exposed to TcdB (10 ng/ml) with or without serum for 1 h followed by Western Blot analysis using a monoclonal antibody that only recognizes non-glucosylated Rac1. β-actin was used as an equal loading control. (B) Quantitation of Rac1 levels in (A) ( ** p

    Techniques Used: Activity Assay, Western Blot, Quantitation Assay

    15) Product Images from "The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome"

    Article Title: The E3 ubiquitin ligase Pellino2 mediates priming of the NLRP3 inflammasome

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03669-z

    Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p
    Figure Legend Snippet: Pellino2 ubiquitinates IRAK1 and suppress the inhibitory effects of IRAK1 on NLRP3. a Immunoblot analysis of myc and IRAK1 in lysates (Input) and immunoprecipitated (IP) myc samples from HEK293T cells transfected with myc-tagged Pellino2 and untagged IRAK1. b Immunoblot analysis of HA, IRAK1 and FLAG in lysates (Input) and IP IRAK1 samples from HEK293T cells transfected with FLAG-tagged Pellino2, FLAG-tagged Pellino2 RING mutant, untagged IRAK1, and HA-Ubiquitin. c, d Immunoblot analysis of Ubiquitin (short exposure (SE) or long exposure (LE)) and IRAK1 in lysates (Input) and IP IRAK1 samples or IRAK1 and ubq in TUBE-Ubq elution and cell lysates ( d ) from WT and Peli2 −/− BMDMs treated with 100 ng/ml of LPS for the indicated times. e Immunoblot analysis of NLRP3 and IRAK1 in lysates (Input) and IP IRAK1 samples from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. f ELISA of IL-1β (left panel) and TNF (right panel) in medium from primary WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for 3 h and then with 2.5 mM ATP or 5 mM Nigericin for 1 h. UT, untreated. g Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates of WT and Irak1 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h and 2.5 mM ATP for 1 h. h Immunoblot analysis of HA, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from HEK293T cells transfected with V5-tagged NLRP3, untagged IRAK1, untagged kinase dead IRAK1 (IRAK1-KD), and HA-ubiquitin. β-Actin was used as loading controls. i Immunoblot analysis of Ubiquitin, NLRP3, and IRAK1 in lysates (Input) and IP NLRP3 samples from immortalized WT and Irak1 −/− BMDMs treated with 100 ng/ml LPS for the indicated times. j Immortalized WT and Irak1 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING) or Pellino2 FHA mutant (FHA). Immunoblot analysis of myc and NLRP3 in lysates (Input) and IP myc samples from virus-infected BMDMs treated with 100 ng/ml LPS for indicated times. * p

    Techniques Used: Immunoprecipitation, Transfection, Mutagenesis, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p
    Figure Legend Snippet: Pellino2 mediates NLRP3-dependent oligomerization of ASC. a Immunofluorescence staining of ASC in WT and Peli2 −/− BMDMs that were left untreated (UT) or treated with 100 ng/ml LPS for 3 h and further stimulated with ATP for 30 min. ASC specks were detected by immunostaining using anti-ASC antibody and anti-rabbit Alexa Fluor 568 (red) and cells were counter stained with nuclei-staining DAPI. The histogram quantitates the percentage of cells that exhibit ASC speck formation. (scale bar = 100 μm). b, c Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further treatment with b 2.5 mM ATP, or c 5 mM Nigericin for 30 min. β-Actin was used as loading controls. d WT and Peli2 −/− BMDMs were infected with MSCV as control (Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (Peli2), Pellino2 RING mutant (RING), or Pellino2 FHA mutant (FHA). Immunoblot analysis of ASC in chemically cross-linked NP-40 insoluble fractions and in NP-40 soluble fractions from cell lysates of MSCV-infected cells treated with 100 ng/ml LPS for 3 h followed by 2.5 mM ATP for 30 min. The expression of the Pellino2 constructs was measured by immunoblotting with an anti-myc antibody. ** p

    Techniques Used: Immunofluorescence, Staining, Immunostaining, Infection, Expressing, Construct, Mutagenesis

    Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p
    Figure Legend Snippet: Pellino2 mediates LPS-induced ubiquitination and activation of NLRP3. a-c Immunoblot analysis of NLRP3 and ubiquitin in cell lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( a ) or NLRP3 and K63-linked ubiquitin (K63-ubq) in K63-TUBE-FLAG elution and cell lysates ( b ) or NLRP3 and K48-ubq in K48-TUBE-FLAG elution and cell lysates ( c ) from WT and Peli2 −/− BMDMs treated with a 100 ng/ml LPS for the indicated times. d ELISA of IL-1β in medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 2 h followed by sequential treatment with 1 μM MCC950 for 1 h and 2.5 mM ATP for 30 min. UT, untreated. e, f Immunoblot analysis of ubiquitin and NLRP3 in lysates (Input) and immunoprecipitated (IP) NLRP3 samples ( e ) or NLRP3 and K63-ubq in K63-TUBE-FLAG elution and cell lysates ( f ) from WT and Peli2 −/− BMDMs pre-treated with 1 μM MCC950 for 1 h followed by treatment with 100 ng/ml LPS for the indicated times. g Peli2 −/− BMDMs were infected with MSCV as control (MSCV-Ctrl) or with MSCV containing an expression construct encoding myc-tagged murine Pellino2 (MSCV-Peli2). Immunoblot analysis of NLRP3 and myc in lysates (Input) and immunoprecipitated (IP) myc samples from MSCV-infected cells treated with 100 ng/ml LPS for the indicated times. β-Actin was used as loading controls. * p

    Techniques Used: Activation Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Infection, Expressing, Construct

    Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p
    Figure Legend Snippet: Pellino2 mediates activation of the NLRP3 pathway. ELISA of a IL-1β and b IL-18, and c LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. UT, untreated. d Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. ELISA of e IL-1β and f IL-18 and g LDH assay of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h. h Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from WT and Peli2 −/− BMDMs stimulated with 100 ng/ml LPS for 3 h with or without further stimulation with 5 mM Nigericin for 1 h or transfection of Poly (dA:dT) (1 μg/ml) for 6 h. i ELISA of IL-1β of medium from WT and Peli2 −/− BMDMs treated with 100 ng/ml LPS for 3 h with or without further stimulation with the indicated concentrations of Alum for 6 h. j ELISA of IL-1β in medium from peritoneal-resident macrophages isolated from WT and Peli2 −/− mice. Cells were treated with 100 ng/ml of LPS for 3 h with or without further stimulation with 2.5 mM ATP for 1 h. k-m Human THP1 cells were transfected with human Pellino2-specific siRNA or control siRNA. k Quantitative RT-PCR analysis of PELI2 expression in transfected cells. l ELISA of IL-1β in medium from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further treatment with 2.5 mM ATP or 5 mM Nigericin for 1 h. m Immunoblot analysis of IL-1β and Caspase-1 in medium (Sup) and lysates from transfected THP1 cells stimulated with 100 ng/ml LPS for 6 h with or without further stimulation with 2.5 mM ATP or 5 mM Nigericin for 1 h. β-Actin was used as loading controls. * p

    Techniques Used: Activation Assay, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, Transfection, Isolation, Mouse Assay, Quantitative RT-PCR, Expressing

    Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p
    Figure Legend Snippet: Pellino2 mediates activation of the NLRP3 pathway in response to bacterial infection. a-f WT and Peli2 −/− BMDMs were infected with a, b C. rodentium , c, d E.coli , or e, f P. aeruginosa (PA01 strain) at a multiplicity of infection (MOI) of 100. a, c, e ELISA of IL-1β, IL-18, and CXCL1 in medium from BMDMs infected for 6 h. b, d Immunoblot analysis of Caspase-11 in lysates from cells infected for 0–6 h. f Immunoblot analysis of IL-1β and Caspase-1 in lysates from cells infected with PAO1 for 3 h. β-Actin was used as loading controls. g ELISA of IL-1β, IL-18, and IL-6 in peritoneal lavage from WT and Peli2 −/− mice previously infected for 10 h by intraperitoneal injection of PAO1 (1.5 × 10 7 CFU). * p

    Techniques Used: Activation Assay, Infection, Enzyme-linked Immunosorbent Assay, Mouse Assay, Injection

    16) Product Images from "Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models *Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models * ♦"

    Article Title: Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models *Diaminothiazoles Modify Tau Phosphorylation and Improve the Tauopathy in Mouse Models * ♦

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.436402

    Tau phosphorylation is reduced by kinase inhibitor diaminothiazole series in vivo . A and B, phosphorylated Tau at 55 kDa detected by PHF-1 is reduced by diaminothiazole treatment in CK-p25 mice. A, representative images of the reduction in PHF-1 signal following treatment with LDN-193594 compared with the vehicle-treated mice (experiments 1, 2, and 4). Western blots of CK-p25 mice did not reveal a PHF-1 65-kDa band. B, ratio of PHF-1 to β-actin densitometry was calculated and normalized to vehicle ( v ). Normalized values are reported as mean ± S.E. n ≥3. C and D, phosphorylated Tau at 65 kDa detected by PHF-1 was specifically reduced by diaminothiazole treatment in 3×Tg-AD mice. C, representative images showing reduction of PHF-1 signal following treatment with LDN-193594 compared with the vehicle-treated mice. Cortex ( left ) and hippocampus ( right ) are shown. The arrow indicates the 65-kDa phospho-Tau band reduced with treatment. D, ratio of PHF-1 to β-actin densitometry was calculated for the 65-kDa band in experiments 6–9, and normalized to vehicle. Normalized values are reported as mean ± S.E. n ≥4. E and F, correlation of PHF-1 + cell numbers with densitometry of 65 kDa ( E ) and 55 kDa ( F ) PHF-1 Tau bands by Western blotting. Statistical significance is indicated: *, p
    Figure Legend Snippet: Tau phosphorylation is reduced by kinase inhibitor diaminothiazole series in vivo . A and B, phosphorylated Tau at 55 kDa detected by PHF-1 is reduced by diaminothiazole treatment in CK-p25 mice. A, representative images of the reduction in PHF-1 signal following treatment with LDN-193594 compared with the vehicle-treated mice (experiments 1, 2, and 4). Western blots of CK-p25 mice did not reveal a PHF-1 65-kDa band. B, ratio of PHF-1 to β-actin densitometry was calculated and normalized to vehicle ( v ). Normalized values are reported as mean ± S.E. n ≥3. C and D, phosphorylated Tau at 65 kDa detected by PHF-1 was specifically reduced by diaminothiazole treatment in 3×Tg-AD mice. C, representative images showing reduction of PHF-1 signal following treatment with LDN-193594 compared with the vehicle-treated mice. Cortex ( left ) and hippocampus ( right ) are shown. The arrow indicates the 65-kDa phospho-Tau band reduced with treatment. D, ratio of PHF-1 to β-actin densitometry was calculated for the 65-kDa band in experiments 6–9, and normalized to vehicle. Normalized values are reported as mean ± S.E. n ≥4. E and F, correlation of PHF-1 + cell numbers with densitometry of 65 kDa ( E ) and 55 kDa ( F ) PHF-1 Tau bands by Western blotting. Statistical significance is indicated: *, p

    Techniques Used: In Vivo, Mouse Assay, Western Blot

    17) Product Images from "Morphological characterization of NG2 glia and their association with neuroglial cells in the 3-nitropropionic acid–lesioned striatum of rat"

    Article Title: Morphological characterization of NG2 glia and their association with neuroglial cells in the 3-nitropropionic acid–lesioned striatum of rat

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-24385-0

    Representative images showing the temporal profiles of NG2-positive cells in 3-NP-treated rat forebrains. ( a ) Low-magnification view of a coronal section from a saline-treated control. ( b ) Higher-magnification view of the boxed area shown in a. Note that weak NG2 expression was observed in small stellate cells with fine processes in control striatum. ( c – e ) Prominent NG2 immunoreactivity appeared at the lesion edge by day 1 ( c ), and appeared to move into the epicenter on days 2 ( d ) and 3 ( e ) post-lesion. The boxed areas in e and g are enlarged in g and h, respectively. Note that NG2-positive cells in the lesion periphery had irregular cell bodies with thick and short processes (arrows in h), while small stellate cells with long fine processes (arrowhead in h) were preferentially found in the epicenter. ( f ) Merged image of FJB staining and GFAP immunoreactivity using serial striatal sections obtained 3 days post-lesion. Note that the lesion core is distinguished by intense FJB staining and concomitant loss of GFAP-positive astrocytes. ( i – k ) Changes in NG2 immunoreactivity in striatal sections from 3-NP-treated rats 7 ( i ), 14 ( j ), and 28 days (k) post-lesion. Prominent NG2 immunoreactivity was localized within both the lesion edge and the epicenter. LV: lateral ventricle. ( l ) Representative western blot analysis results for NG2 protein expression in striatal extracts from sham controls and rats killed 7, 14, and 28 days post-lesion. Note that a band of about 300 kDa corresponding to NG2 protein was clearly observed in both the control and lesioned striatum. ( m ) Quantification of NG2 protein expression. Data were obtained using densitometry and were normalized using β-actin as the loading control. The results are expressed in relative optical density and represent means ± SEM. The intensity of NG2 protein reactivity significantly increased by 14 days after 3-NP injection and then decreased, although enhanced expression levels persisted until at least day 28. **P
    Figure Legend Snippet: Representative images showing the temporal profiles of NG2-positive cells in 3-NP-treated rat forebrains. ( a ) Low-magnification view of a coronal section from a saline-treated control. ( b ) Higher-magnification view of the boxed area shown in a. Note that weak NG2 expression was observed in small stellate cells with fine processes in control striatum. ( c – e ) Prominent NG2 immunoreactivity appeared at the lesion edge by day 1 ( c ), and appeared to move into the epicenter on days 2 ( d ) and 3 ( e ) post-lesion. The boxed areas in e and g are enlarged in g and h, respectively. Note that NG2-positive cells in the lesion periphery had irregular cell bodies with thick and short processes (arrows in h), while small stellate cells with long fine processes (arrowhead in h) were preferentially found in the epicenter. ( f ) Merged image of FJB staining and GFAP immunoreactivity using serial striatal sections obtained 3 days post-lesion. Note that the lesion core is distinguished by intense FJB staining and concomitant loss of GFAP-positive astrocytes. ( i – k ) Changes in NG2 immunoreactivity in striatal sections from 3-NP-treated rats 7 ( i ), 14 ( j ), and 28 days (k) post-lesion. Prominent NG2 immunoreactivity was localized within both the lesion edge and the epicenter. LV: lateral ventricle. ( l ) Representative western blot analysis results for NG2 protein expression in striatal extracts from sham controls and rats killed 7, 14, and 28 days post-lesion. Note that a band of about 300 kDa corresponding to NG2 protein was clearly observed in both the control and lesioned striatum. ( m ) Quantification of NG2 protein expression. Data were obtained using densitometry and were normalized using β-actin as the loading control. The results are expressed in relative optical density and represent means ± SEM. The intensity of NG2 protein reactivity significantly increased by 14 days after 3-NP injection and then decreased, although enhanced expression levels persisted until at least day 28. **P

    Techniques Used: Expressing, Staining, Western Blot, Injection

    18) Product Images from "Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates"

    Article Title: Mechanisms underlying extensive Ser129-phosphorylation in α-synuclein aggregates

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-017-0452-6

    Relation of Ser129-phosphorylated α-syn levels to total α-syn ones in intra- and extracellular spaces. CHO cells were transfected with the empty vector or the indicated amounts of wild-type α-syn cDNA. Samples were loaded along with recombinant α-syn proteins and Ser129-phosphorylated α-syn proteins for standards, followed by western blotting. Bands of Ser129-phosphorylated α-syn and total α-syn, including phosphorylated and non-phosphorylated forms, were detected by EP1536Y and Syn-1 antibody, respectively. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were corrected by plotting them on the standard curves, and then normalized to the intensities of β-actin. a Relation in intracellular α-syn. Cell lystaes (10 μg/lane) were loaded. b Relation in extracellular α-syn. After TCA-precipitated proteins were resolved by Laemmli’s sample buffer, samples corresponding to 20% of CM volume were loaded. Graphs show the positive correlation between Ser-129 phosphorylated and total α-syn levels
    Figure Legend Snippet: Relation of Ser129-phosphorylated α-syn levels to total α-syn ones in intra- and extracellular spaces. CHO cells were transfected with the empty vector or the indicated amounts of wild-type α-syn cDNA. Samples were loaded along with recombinant α-syn proteins and Ser129-phosphorylated α-syn proteins for standards, followed by western blotting. Bands of Ser129-phosphorylated α-syn and total α-syn, including phosphorylated and non-phosphorylated forms, were detected by EP1536Y and Syn-1 antibody, respectively. Relative band intensities of Ser129-phosphorylated α-syn and total α-syn were corrected by plotting them on the standard curves, and then normalized to the intensities of β-actin. a Relation in intracellular α-syn. Cell lystaes (10 μg/lane) were loaded. b Relation in extracellular α-syn. After TCA-precipitated proteins were resolved by Laemmli’s sample buffer, samples corresponding to 20% of CM volume were loaded. Graphs show the positive correlation between Ser-129 phosphorylated and total α-syn levels

    Techniques Used: Transfection, Plasmid Preparation, Recombinant, Western Blot

    Effects of Ca 2+ on Ser129-phosphorylation of α-syn. SH-SY5Y cell lines stably expressing wild-type α-syn (wt-aS/SH #4) were incubated in media containing 5 μM calcium ionophore A23187 except b . As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (15 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or anti-β-actin (AC-15) antibody. a Effect of A23187 incubation time on Ser129-phosphorylation. Cells were treated with A23187 for the indicated time points until 8 h. b Effect of A23187 concentrations on Ser129-phosphorylation. Cells were treated with A23187 at the indicated concentrations for 8 h. c, d Effect of extracellular Ca 2+ chelator EGTA ( c) or intracellular Ca 2+ chelator BAPTA-AM (B-AM) ( d ) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 5 μM A23187 with the indicated concentrations of EGTA or BAPTA-AM for 4 h. e, f Effect of CaM inhibitor W-7 ( e ) or calmidazolium (Calm) ( f ) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing A23187 with the indicated concentrations of W-7 or calmidazolium for 4 h. Representative blots are shown. In the graphs of a to f , relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P
    Figure Legend Snippet: Effects of Ca 2+ on Ser129-phosphorylation of α-syn. SH-SY5Y cell lines stably expressing wild-type α-syn (wt-aS/SH #4) were incubated in media containing 5 μM calcium ionophore A23187 except b . As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (15 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or anti-β-actin (AC-15) antibody. a Effect of A23187 incubation time on Ser129-phosphorylation. Cells were treated with A23187 for the indicated time points until 8 h. b Effect of A23187 concentrations on Ser129-phosphorylation. Cells were treated with A23187 at the indicated concentrations for 8 h. c, d Effect of extracellular Ca 2+ chelator EGTA ( c) or intracellular Ca 2+ chelator BAPTA-AM (B-AM) ( d ) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing 5 μM A23187 with the indicated concentrations of EGTA or BAPTA-AM for 4 h. e, f Effect of CaM inhibitor W-7 ( e ) or calmidazolium (Calm) ( f ) on A23187-induced Ser129-phosphorylation. Cells were incubated in media containing A23187 with the indicated concentrations of W-7 or calmidazolium for 4 h. Representative blots are shown. In the graphs of a to f , relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P

    Techniques Used: Stable Transfection, Expressing, Incubation, Concentration Assay, SDS Page, Western Blot, Chick Chorioallantoic Membrane Assay

    Role of Ca 2+ and CaM in mitochondria complex I inhibitor-induced Ser129-phosphorylation of α-syn. As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a, b Effect of intracellular Ca 2+ chelator BAPTA-AM ( a ) or extracelluar Ca 2+ chelator EGTA ( b ) on MPP + -induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 1 mM MPP + and the indicated concentrations of BAPTA-AM or EGTA for 16 h. c, d Effect of BAPTA-AM ( c ) and EGTA ( d ) on rotenone-induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 5 μM rotenone and the indicated concentrations of BAPTA-AM or EGTA for 16 h. e, f Effect of CaM inhibitor W-7 on MPP + ( e )- or rotenone ( f )-induced Ser129-phosphorylation. Cells were incubated in media containing 1 mM MPP + or 5 μM rotenone with W-7 at the indicated concentrations for 16 h. Representative blots are shown. In the graphs of a to f , relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P
    Figure Legend Snippet: Role of Ca 2+ and CaM in mitochondria complex I inhibitor-induced Ser129-phosphorylation of α-syn. As vehicle control, cells were treated with DMSO at the same final concentration as reagents used. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a, b Effect of intracellular Ca 2+ chelator BAPTA-AM ( a ) or extracelluar Ca 2+ chelator EGTA ( b ) on MPP + -induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 1 mM MPP + and the indicated concentrations of BAPTA-AM or EGTA for 16 h. c, d Effect of BAPTA-AM ( c ) and EGTA ( d ) on rotenone-induced Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 5 μM rotenone and the indicated concentrations of BAPTA-AM or EGTA for 16 h. e, f Effect of CaM inhibitor W-7 on MPP + ( e )- or rotenone ( f )-induced Ser129-phosphorylation. Cells were incubated in media containing 1 mM MPP + or 5 μM rotenone with W-7 at the indicated concentrations for 16 h. Representative blots are shown. In the graphs of a to f , relative band intensities of Ser129-phosphorylated α-syn and total α-syn were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction (*, P

    Techniques Used: Chick Chorioallantoic Membrane Assay, Concentration Assay, SDS Page, Western Blot, Incubation

    Effects of mitochondrial complex I inhibitors MPP + and rotenone on Ser129-phosphorylation of α-syn. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Effect of incubation time of MPP + and rotenone on Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 1 mM MPP + or 5 μM rotenone for the indicated time points until 16 h. Vehicle controls were treated with DMSO at the same final concentration as each reagent. b Effect of concentrations of MPP + or rotenone on Ser129-phosphorylation. Cells were incubated in media containing the indicated amounts of MPP + or rotenone for 12 h. Graphs show relative band intensities of Ser129-phosphorylated α-syn and total α-syn. They were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P
    Figure Legend Snippet: Effects of mitochondrial complex I inhibitors MPP + and rotenone on Ser129-phosphorylation of α-syn. Cell lysates (10 μg/lane) were loaded on SDS-PAGE and analyzed by western botting with EP1536Y, Syn-1, or AC-15 antibody. a Effect of incubation time of MPP + and rotenone on Ser129-phosphorylation. Wt-aS/SH #4 cells were incubated in media containing 1 mM MPP + or 5 μM rotenone for the indicated time points until 16 h. Vehicle controls were treated with DMSO at the same final concentration as each reagent. b Effect of concentrations of MPP + or rotenone on Ser129-phosphorylation. Cells were incubated in media containing the indicated amounts of MPP + or rotenone for 12 h. Graphs show relative band intensities of Ser129-phosphorylated α-syn and total α-syn. They were normalized to those of β-actin. Data represent means ± SD and P values were estimated by one-way ANOVA with Bonferroni correction or Welch-ANOVA with Games-Howell post hoc test for unequal-variances (*, P

    Techniques Used: SDS Page, Western Blot, Incubation, Concentration Assay

    19) Product Images from "Kinase Activity of Fibroblast Growth Factor Receptor 3 Regulates Activity of the Papillomavirus E2 Protein"

    Article Title: Kinase Activity of Fibroblast Growth Factor Receptor 3 Regulates Activity of the Papillomavirus E2 Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.01066-17

    FGFR3 interacts with PV E2 but not with EGFR or EphB2. (A) HEK293TT cells were transfected with FGFR3 WT, K650E, and K508R constructs along with a BPV-1 E2 expression plasmid. BPV-1 E2 pulldown was completed with B201/B202 antibodies and blotted with FGFR3, B201, and β-actin antibodies. (B) HEK293TT cells were transfected with FGFR3 WT, K650E, and K508R constructs along with a FLAG-HPV-31 E2 expression plasmid. HPV-31 E2 pulldown was completed with M2 antibodies and blotted with FGFR3, M2, and β-actin antibodies. C33A cells were transfected with wild-type BPV-1 E2, E2R, and EGFR (C) or EphB2 (D) expression plasmids. Twenty-four hours later, cells were lysed, and BPV-1 E2 was immunoprecipitated with B201 antibodies. Immunoblotting was completed with EGFR, EphB2, and B201 antibodies. IP, immunoprecipitation.
    Figure Legend Snippet: FGFR3 interacts with PV E2 but not with EGFR or EphB2. (A) HEK293TT cells were transfected with FGFR3 WT, K650E, and K508R constructs along with a BPV-1 E2 expression plasmid. BPV-1 E2 pulldown was completed with B201/B202 antibodies and blotted with FGFR3, B201, and β-actin antibodies. (B) HEK293TT cells were transfected with FGFR3 WT, K650E, and K508R constructs along with a FLAG-HPV-31 E2 expression plasmid. HPV-31 E2 pulldown was completed with M2 antibodies and blotted with FGFR3, M2, and β-actin antibodies. C33A cells were transfected with wild-type BPV-1 E2, E2R, and EGFR (C) or EphB2 (D) expression plasmids. Twenty-four hours later, cells were lysed, and BPV-1 E2 was immunoprecipitated with B201 antibodies. Immunoblotting was completed with EGFR, EphB2, and B201 antibodies. IP, immunoprecipitation.

    Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Immunoprecipitation

    Activated FGFR3 increases HPV-31 E2 levels. HEK293TT cells were transfected with pCI or FLAG-FGFR3 K650E and FLAG-HPV-31 E1 or FLAG-HPV-31 E2 constructs. Forty-eight hours later, cells were lysed in 2% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0. Immunoblotting was completed with FGFR3, M2 (FLAG), and β-actin antibodies.
    Figure Legend Snippet: Activated FGFR3 increases HPV-31 E2 levels. HEK293TT cells were transfected with pCI or FLAG-FGFR3 K650E and FLAG-HPV-31 E1 or FLAG-HPV-31 E2 constructs. Forty-eight hours later, cells were lysed in 2% SDS, 150 mM NaCl, 10 mM Tris-HCl, pH 8.0. Immunoblotting was completed with FGFR3, M2 (FLAG), and β-actin antibodies.

    Techniques Used: Transfection, Construct

    FGFR3 siRNA increases HPV-31 DNA content in CIN612-9E cells. (A) CIN612-9E cells were transfected with a control, FGFR3, or EGFR siRNA. After 48 h cells were lysed (0.5% NP-40), and immunoblotting was completed with FGFR3, EGFR, and β-actin antibodies. (B) CIN612-9E cells were transfected with a control, FGFR3, or EGFR siRNA. After 48 h cells were lysed, and DNA was isolated for reverse transcription-PCR against the HPV-31 LCR and normalized to the level with the β-actin primer set. Values are expressed as means ± SEM ( n = 14). *, P
    Figure Legend Snippet: FGFR3 siRNA increases HPV-31 DNA content in CIN612-9E cells. (A) CIN612-9E cells were transfected with a control, FGFR3, or EGFR siRNA. After 48 h cells were lysed (0.5% NP-40), and immunoblotting was completed with FGFR3, EGFR, and β-actin antibodies. (B) CIN612-9E cells were transfected with a control, FGFR3, or EGFR siRNA. After 48 h cells were lysed, and DNA was isolated for reverse transcription-PCR against the HPV-31 LCR and normalized to the level with the β-actin primer set. Values are expressed as means ± SEM ( n = 14). *, P

    Techniques Used: Transfection, Isolation, Polymerase Chain Reaction

    20) Product Images from "Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis *"

    Article Title: Printor, a Novel TorsinA-interacting Protein Implicated in Dystonia Pathogenesis *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.004838

    Printor co-distributes with torsinA in multiple tissues and brain regions. A , lysates from transfected SH-SY5Y cells expressing Myc-tagged printor or purified His-tagged printor protein were analyzed by immunoblotting with preimmune serum, anti-printor antibody, anti-printor antibody preabsorbed with recombinant printor protein, or anti-Myc antibody. Recomb. , recombinant. B , equal amounts of lysates (50 μg) from the indicated cells were analyzed by immunoblotting using anti-printor, preabsorbed anti-printor, and anti-β-actin antibodies. The asterisks indicate bands that probably represent printor degradation products. C , equal amounts of homogenates (100 μg) from the indicated rat tissues were analyzed by immunoblotting using anti-printor, anti-torsinA, and anti-β-actin antibodies. Sk. , skeletal. D , equal amounts of homogenates (100 μg) from the indicated rat brain regions were analyzed by immunoblotting using anti-printor, anti-torsinA, and anti-β-actin antibodies. Sup. , superior; Inf. , inferior. E , SH-SY5Y cells overexpressing Myc-tagged printor ( top ) were immunostained with primary antibodies against printor and the Myc tag, followed by detection with secondary antibodies conjugated to Texas Red (Myc, red ) or FITC (printor, green ). Primary cortical neurons ( bottom ) were immunostained with primary antibodies against printor and MAP2, followed by detection with secondary antibodies conjugated to Texas Red (printor, red ) or FITC (MAP2, green ). Hoechst stain was used to visualize the nucleus. Scale bar , 10 μm. All data are representative of at least three independent experiments.
    Figure Legend Snippet: Printor co-distributes with torsinA in multiple tissues and brain regions. A , lysates from transfected SH-SY5Y cells expressing Myc-tagged printor or purified His-tagged printor protein were analyzed by immunoblotting with preimmune serum, anti-printor antibody, anti-printor antibody preabsorbed with recombinant printor protein, or anti-Myc antibody. Recomb. , recombinant. B , equal amounts of lysates (50 μg) from the indicated cells were analyzed by immunoblotting using anti-printor, preabsorbed anti-printor, and anti-β-actin antibodies. The asterisks indicate bands that probably represent printor degradation products. C , equal amounts of homogenates (100 μg) from the indicated rat tissues were analyzed by immunoblotting using anti-printor, anti-torsinA, and anti-β-actin antibodies. Sk. , skeletal. D , equal amounts of homogenates (100 μg) from the indicated rat brain regions were analyzed by immunoblotting using anti-printor, anti-torsinA, and anti-β-actin antibodies. Sup. , superior; Inf. , inferior. E , SH-SY5Y cells overexpressing Myc-tagged printor ( top ) were immunostained with primary antibodies against printor and the Myc tag, followed by detection with secondary antibodies conjugated to Texas Red (Myc, red ) or FITC (printor, green ). Primary cortical neurons ( bottom ) were immunostained with primary antibodies against printor and MAP2, followed by detection with secondary antibodies conjugated to Texas Red (printor, red ) or FITC (MAP2, green ). Hoechst stain was used to visualize the nucleus. Scale bar , 10 μm. All data are representative of at least three independent experiments.

    Techniques Used: Transfection, Expressing, Purification, Recombinant, Staining

    21) Product Images from "Regulation of Prostate Cancer Progression by Galectin-3"

    Article Title: Regulation of Prostate Cancer Progression by Galectin-3

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2009.080816

    Up-regulation and nuclear transport of p21 in galectin-3 knockdown clones. A: Top: Galectin-3 knockdown increased p21 expression and suppressed phosphorylation of pRb in total cell lysate. β-Actin was used as the loading control. A: Bottom: Quantification
    Figure Legend Snippet: Up-regulation and nuclear transport of p21 in galectin-3 knockdown clones. A: Top: Galectin-3 knockdown increased p21 expression and suppressed phosphorylation of pRb in total cell lysate. β-Actin was used as the loading control. A: Bottom: Quantification

    Techniques Used: Clone Assay, Expressing

    22) Product Images from "Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes"

    Article Title: Low paternal dietary folate alters the mouse sperm epigenome and is associated with negative pregnancy outcomes

    Journal: Nature Communications

    doi: 10.1038/ncomms3889

    Differential gene expression in placenta of offspring sired by FD versus FS males. ( a ) Heat-map showing the expression levels of 39 genes in four placentas of 18.5 dpc fetuses sired by either an FS ( n =4) or FD ( n =4) male. Placentas analysed were from unique litters. ( b ) Validation of array results by real-time PCR on an extended group of samples ( n =8, FS and n =8, FD). ( c ) Selected array targets Cav1 and Txndc16 showed altered gene expression and were differentially methylated in sperm of FD sires. Data are expressed as a percentage of the control β-actin, with the value of the FS at 100%. Means±s.e.m. of eight determinations are shown. For b and c , * P
    Figure Legend Snippet: Differential gene expression in placenta of offspring sired by FD versus FS males. ( a ) Heat-map showing the expression levels of 39 genes in four placentas of 18.5 dpc fetuses sired by either an FS ( n =4) or FD ( n =4) male. Placentas analysed were from unique litters. ( b ) Validation of array results by real-time PCR on an extended group of samples ( n =8, FS and n =8, FD). ( c ) Selected array targets Cav1 and Txndc16 showed altered gene expression and were differentially methylated in sperm of FD sires. Data are expressed as a percentage of the control β-actin, with the value of the FS at 100%. Means±s.e.m. of eight determinations are shown. For b and c , * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Methylation

    23) Product Images from "A Novel KCNJ2 Mutation Identified in an Autistic Proband Affects the Single Channel Properties of Kir2.1"

    Article Title: A Novel KCNJ2 Mutation Identified in an Autistic Proband Affects the Single Channel Properties of Kir2.1

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2018.00076

    Expression level of WT and p.Phe58Ser channel. (A) Immunoblotting analysis on total protein extracts from untransfected tsA201 cells (NT), tsA201 cells transfected with pcDNA3.1-NT-GFP-TOPO-hKCNJ2-WT plasmid (WT) or with pcDNA3.1-NT-GFP-TOPO-hKCNJ2-p.Phe58Ser plasmid (p.Phe58Ser) is showed. β-Actin was used as endogenous controls of equal protein load. (B) Densitometry analysis of Kir2.1 expression levels in total protein extracts from tsA201 cell lines. Data are expressed as fold change ratio on untransfected cells (NT) where p.Phe58Ser and WT indicate the relative protein expression in cells transfected with the mutant or WT encoding plasmids, respectively, normalized to the β-Actin protein expression levels. Data shown are representative of five independent experiments.
    Figure Legend Snippet: Expression level of WT and p.Phe58Ser channel. (A) Immunoblotting analysis on total protein extracts from untransfected tsA201 cells (NT), tsA201 cells transfected with pcDNA3.1-NT-GFP-TOPO-hKCNJ2-WT plasmid (WT) or with pcDNA3.1-NT-GFP-TOPO-hKCNJ2-p.Phe58Ser plasmid (p.Phe58Ser) is showed. β-Actin was used as endogenous controls of equal protein load. (B) Densitometry analysis of Kir2.1 expression levels in total protein extracts from tsA201 cell lines. Data are expressed as fold change ratio on untransfected cells (NT) where p.Phe58Ser and WT indicate the relative protein expression in cells transfected with the mutant or WT encoding plasmids, respectively, normalized to the β-Actin protein expression levels. Data shown are representative of five independent experiments.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Mutagenesis

    24) Product Images from "Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation"

    Article Title: Epac–protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation

    Journal: Pain

    doi: 10.1097/j.pain.0000000000000547

    Epac1 and Epac2 expression and cyclic adenosine monophosphate (cAMP) level increase after complete Freund adjuvant (CFA)–induced inflammation. (A) Expression of Epac1 and Epac2 in control (Con) and CFA-treated rats. Western blots of Epac1 and Epac2 expressed in L4 and L5 dorsal root ganglia (DRGs) isolated from control rat 1 and 2 or from inflamed rat 3 to 6. Same rat protein samples were used for both Epac1 and Epac2 measurements. (B) Summary of Epac1 and Epac2 expression. The intensity of each protein band was determined and normalized with its respective β-actin, which was used as a loading control. Relative protein intensity after CFA is expressed as fold-increase by dividing the normalized protein intensity obtained after CFA with respect to that obtained under pre-CFA control condition. Compared to the control, Epac1 expression after CFA was (CFA/Con = 3.5 ± 0.5; n = 4) and Epac2 expression after CFA was (CFA/Con = 3.0 ± 0.6; n = 4). * P
    Figure Legend Snippet: Epac1 and Epac2 expression and cyclic adenosine monophosphate (cAMP) level increase after complete Freund adjuvant (CFA)–induced inflammation. (A) Expression of Epac1 and Epac2 in control (Con) and CFA-treated rats. Western blots of Epac1 and Epac2 expressed in L4 and L5 dorsal root ganglia (DRGs) isolated from control rat 1 and 2 or from inflamed rat 3 to 6. Same rat protein samples were used for both Epac1 and Epac2 measurements. (B) Summary of Epac1 and Epac2 expression. The intensity of each protein band was determined and normalized with its respective β-actin, which was used as a loading control. Relative protein intensity after CFA is expressed as fold-increase by dividing the normalized protein intensity obtained after CFA with respect to that obtained under pre-CFA control condition. Compared to the control, Epac1 expression after CFA was (CFA/Con = 3.5 ± 0.5; n = 4) and Epac2 expression after CFA was (CFA/Con = 3.0 ± 0.6; n = 4). * P

    Techniques Used: Expressing, Western Blot, Isolation

    25) Product Images from "Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons"

    Article Title: Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons

    Journal: Journal of Virology

    doi: 10.1128/JVI.03129-14

    Comparison HCV NS3-4A-dependent cleavage of and interference with hMAVS and mMAVS in human cells. (A and B) Appearance of human (A) or mouse (B) MAVS expression in the presence of HCV protease in human HEK 293T-MAVS −/− miR122 cells. The cells were reverse transfected with reporter plasmids (100 ng) together with vector expressing each indicated gene (100 ng). A day after transfection, the medium was changed and the indicated cells were treated with addition of 10 μM boceprevir, a protease inhibitor (P.I). Twenty-four hours later, the cells were lysed and specific Western blotting detection using the indicated antibodies was performed. The cleaved (Δ) products of hMAVS and mMAVS are indicated, and β-actin expression was used as a loading control. Images are representative of two individual experiments. (C and D) Effect of HCV protease presence on human (C) or mouse (D) MAVS-dependent IFN-β promoter regulation. HEK 293T-MAVS −/− miR122 cells were reverse transfected with reporter plasmids (100 ng) together with the each indicated expression vector (100 ng). A day after the cells were reverse transfected, the medium was changed and the indicated cells were transfected with either water (mock) or 10 μg/ml of poly(I·C) for 4 h. After that, the cells were treated with addition of water or 10 μM P.I. The cells were then lysed and assayed for luciferase activities 24 h later. Values were then normalized to those for MAVS-GFP-transfected cells. Asterisks show a significant difference between the indicated data. All graph data are shown as mean values ± SDs from three independent experiments, and Western blotting images are representative of two individual experiments.
    Figure Legend Snippet: Comparison HCV NS3-4A-dependent cleavage of and interference with hMAVS and mMAVS in human cells. (A and B) Appearance of human (A) or mouse (B) MAVS expression in the presence of HCV protease in human HEK 293T-MAVS −/− miR122 cells. The cells were reverse transfected with reporter plasmids (100 ng) together with vector expressing each indicated gene (100 ng). A day after transfection, the medium was changed and the indicated cells were treated with addition of 10 μM boceprevir, a protease inhibitor (P.I). Twenty-four hours later, the cells were lysed and specific Western blotting detection using the indicated antibodies was performed. The cleaved (Δ) products of hMAVS and mMAVS are indicated, and β-actin expression was used as a loading control. Images are representative of two individual experiments. (C and D) Effect of HCV protease presence on human (C) or mouse (D) MAVS-dependent IFN-β promoter regulation. HEK 293T-MAVS −/− miR122 cells were reverse transfected with reporter plasmids (100 ng) together with the each indicated expression vector (100 ng). A day after the cells were reverse transfected, the medium was changed and the indicated cells were transfected with either water (mock) or 10 μg/ml of poly(I·C) for 4 h. After that, the cells were treated with addition of water or 10 μM P.I. The cells were then lysed and assayed for luciferase activities 24 h later. Values were then normalized to those for MAVS-GFP-transfected cells. Asterisks show a significant difference between the indicated data. All graph data are shown as mean values ± SDs from three independent experiments, and Western blotting images are representative of two individual experiments.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Protease Inhibitor, Western Blot, Luciferase

    Comparison HCV NS3-4A-dependent cleavage of and interference with hMAVS and mMAVS in mouse cells. (A and B) Human (A) or mouse (B) MAVS was expressed in MLT-MAVS −/− miR122 cells in the presence or absence of GFP or the HCV NS3-4A protease. Expression of proteins was monitored by Western blotting using GFP-, actin-, mMAVS-, or hMAVS-specific antibodies. One day after lentiviral gene transduction, the medium was changed and the indicated cells were treated with addition of 10 μM boceprevir, a protease inhibitor (P.I). Twenty-four hours later, the cells were lysed for specific Western blot analysis. The cleaved (Δ) products of hMAVS and mMAVS are indicated, and β-actin expression was used as a loading control. Images are representative of two individual experiments. (C and D) Effect of HCV NS3-4A protease expression on human (C) or mouse (D) MAVS-dependent type I IFN induction. One day after the cells were transduced, the cells were either mock transfected or transfected with 10 μg/ml of poly(I·C). Four hours later, total RNA was extracted and the relative levels of mIFN-β mRNA expression were determined by qRT-PCR. Data were normalized to the mGAPDH mRNA data. Asterisks show a significant difference between the indicated data. Mean values ± SDs from three independent experiments are given.
    Figure Legend Snippet: Comparison HCV NS3-4A-dependent cleavage of and interference with hMAVS and mMAVS in mouse cells. (A and B) Human (A) or mouse (B) MAVS was expressed in MLT-MAVS −/− miR122 cells in the presence or absence of GFP or the HCV NS3-4A protease. Expression of proteins was monitored by Western blotting using GFP-, actin-, mMAVS-, or hMAVS-specific antibodies. One day after lentiviral gene transduction, the medium was changed and the indicated cells were treated with addition of 10 μM boceprevir, a protease inhibitor (P.I). Twenty-four hours later, the cells were lysed for specific Western blot analysis. The cleaved (Δ) products of hMAVS and mMAVS are indicated, and β-actin expression was used as a loading control. Images are representative of two individual experiments. (C and D) Effect of HCV NS3-4A protease expression on human (C) or mouse (D) MAVS-dependent type I IFN induction. One day after the cells were transduced, the cells were either mock transfected or transfected with 10 μg/ml of poly(I·C). Four hours later, total RNA was extracted and the relative levels of mIFN-β mRNA expression were determined by qRT-PCR. Data were normalized to the mGAPDH mRNA data. Asterisks show a significant difference between the indicated data. Mean values ± SDs from three independent experiments are given.

    Techniques Used: Expressing, Western Blot, Transduction, Protease Inhibitor, Transfection, Quantitative RT-PCR

    mMAVS suppresses HCV replication in transfected mouse liver-derived tumor (MLT) cells. (A) Detection of mMAVS and hMAVS proteins in given human and murine cell lines using MAVS-specific antibodies. Detection of β-actin expression was used as a loading control. (B) Responsiveness of MLT-MAVS −/− miR122 cells with or without mMAVS expression to transfection of poly(I·C). Hep56.1D mouse cells expressing endogenous levels of mMAVS served as a control. A day after seeding, the indicated cells were either left untreated, mock transfected, or transfected with 10 μg/ml of poly(I·C). Four hours later, total cellular RNA was collected and the expression of mIFN-β mRNA was determined by RT-PCR. Expression of IFN-β mRNA was normalized to endogenous levels of mGAPDH and is given relative to the basal expression in Hep56.1D cells. Asterisks show a significant difference between the indicated data. (C) Transient replication of HCV-JFH1 subgenomic replicon with luciferase reporter with an active NS5B polymerase (HCV-SGR) or a ΔGDD polymerase mutant [HCV-SGR (−) Polymerase] in given cell lines. Transfected cells were harvested at the indicated time points, and HCV RNA replication was determined by luciferase assays. RLU, relative light units; h.p.t, hours posttransfection. (D) Detection of HCV NS5A expression (red) in transfected cells by indirect immunofluorescence (IF) analysis 48 h posttransfection. Nuclei were stained with DAPI (blue). All graph data are shown as mean values ± SDs from three independent experiments, and images are representative of three individual experiments.
    Figure Legend Snippet: mMAVS suppresses HCV replication in transfected mouse liver-derived tumor (MLT) cells. (A) Detection of mMAVS and hMAVS proteins in given human and murine cell lines using MAVS-specific antibodies. Detection of β-actin expression was used as a loading control. (B) Responsiveness of MLT-MAVS −/− miR122 cells with or without mMAVS expression to transfection of poly(I·C). Hep56.1D mouse cells expressing endogenous levels of mMAVS served as a control. A day after seeding, the indicated cells were either left untreated, mock transfected, or transfected with 10 μg/ml of poly(I·C). Four hours later, total cellular RNA was collected and the expression of mIFN-β mRNA was determined by RT-PCR. Expression of IFN-β mRNA was normalized to endogenous levels of mGAPDH and is given relative to the basal expression in Hep56.1D cells. Asterisks show a significant difference between the indicated data. (C) Transient replication of HCV-JFH1 subgenomic replicon with luciferase reporter with an active NS5B polymerase (HCV-SGR) or a ΔGDD polymerase mutant [HCV-SGR (−) Polymerase] in given cell lines. Transfected cells were harvested at the indicated time points, and HCV RNA replication was determined by luciferase assays. RLU, relative light units; h.p.t, hours posttransfection. (D) Detection of HCV NS5A expression (red) in transfected cells by indirect immunofluorescence (IF) analysis 48 h posttransfection. Nuclei were stained with DAPI (blue). All graph data are shown as mean values ± SDs from three independent experiments, and images are representative of three individual experiments.

    Techniques Used: Transfection, Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Luciferase, Mutagenesis, Immunofluorescence, Staining

    26) Product Images from "Arginine methylation of SKN-1 promotes oxidative stress resistance in Caenorhabditis elegans"

    Article Title: Arginine methylation of SKN-1 promotes oxidative stress resistance in Caenorhabditis elegans

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101111

    Oxidative stress enhances binding of PRMT-1 to SKN-1, and elevates asymmetrical dimethylation of arginines on SKN-1. (A) GST-PRMT-1 bound to SKN-1 proteins expressed in wild-type worms. GST pull-down assay was performed by immunoprecipitation with GST-PRMT-1 protein from the whole extracts of N2[SKN-1B/C::GFP] worms, followed by immunoblotting with anti-GFP antibody. The arrow on the right indicated the band of GST-PRMT-1 purifed from E. coli BL21. GST protein was as the negative control. Coomassie brilliant blue (CBB) staining (lower) was used as a loading control. (B) SKN-1 interacted with PRMT-1 and was asymmetrically dimethylated on arginines in vivo . co-IP was operated by utilizing the whole extracts of N2[SKN-1B/C::GFP] worms with anti-GFP antibody, followed by immunoblotting with anti-PRMT-1 and anti-ASYM antibody. No antibody was used as the negative control. (C) Oxidative stress increased the binding of PRMT-1 to SKN-1, and elevated asymmetrical dimethylation of arginines on SKN-1. N2[SKN-1B/C::GFP] worms were treated with (or without) 5 mM tBHP, followed by co-IP as the legend to B. β-actin and GFP were utilized as internal references. IgG was used as the negative control.
    Figure Legend Snippet: Oxidative stress enhances binding of PRMT-1 to SKN-1, and elevates asymmetrical dimethylation of arginines on SKN-1. (A) GST-PRMT-1 bound to SKN-1 proteins expressed in wild-type worms. GST pull-down assay was performed by immunoprecipitation with GST-PRMT-1 protein from the whole extracts of N2[SKN-1B/C::GFP] worms, followed by immunoblotting with anti-GFP antibody. The arrow on the right indicated the band of GST-PRMT-1 purifed from E. coli BL21. GST protein was as the negative control. Coomassie brilliant blue (CBB) staining (lower) was used as a loading control. (B) SKN-1 interacted with PRMT-1 and was asymmetrically dimethylated on arginines in vivo . co-IP was operated by utilizing the whole extracts of N2[SKN-1B/C::GFP] worms with anti-GFP antibody, followed by immunoblotting with anti-PRMT-1 and anti-ASYM antibody. No antibody was used as the negative control. (C) Oxidative stress increased the binding of PRMT-1 to SKN-1, and elevated asymmetrical dimethylation of arginines on SKN-1. N2[SKN-1B/C::GFP] worms were treated with (or without) 5 mM tBHP, followed by co-IP as the legend to B. β-actin and GFP were utilized as internal references. IgG was used as the negative control.

    Techniques Used: Binding Assay, Pull Down Assay, Immunoprecipitation, Negative Control, Staining, In Vivo, Co-Immunoprecipitation Assay

    27) Product Images from "RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage"

    Article Title: RFWD3-Mdm2 ubiquitin ligase complex positively regulates p53 stability in response to DNA damage

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0912094107

    RFWD3 is a positive regulator of p53 abundance and regulates the G 1 checkpoint in response to IR. ( A ) U2OS cells were transfected with a RFWD3 siRNA (sequence no. 2) or control siRNA. At 48 h posttransfection, cells were irradiated with 10 Gy IR. Cell lysates were immunoblotted with indicated antibodies. Quantification of relative p53 levels was normalized by β-actin signals. ( B ) The same experiment as in A was performed in MCF7 cells by using a different RFWD3 siRNA (sequence no. 1). ( C ) MCF7 cells were transfected with RFWD3 siRNAs (no. 1 or 2) or a control siRNA. At 48 h posttransfection, cells were irradiated with 2.5 Gy IR. The G 1 checkpoint was measured by the relative S-phase entry at indicated time frames. ( D ) Rescue of the G 1 checkpoint by expressing an RNAi-resistant RFWD3 in U2OS stable shRNA knockdown cells. The cells were transfected with the indicated plasmids and were irradiated with 2.5 Gy IR at 48 hr posttransfection.
    Figure Legend Snippet: RFWD3 is a positive regulator of p53 abundance and regulates the G 1 checkpoint in response to IR. ( A ) U2OS cells were transfected with a RFWD3 siRNA (sequence no. 2) or control siRNA. At 48 h posttransfection, cells were irradiated with 10 Gy IR. Cell lysates were immunoblotted with indicated antibodies. Quantification of relative p53 levels was normalized by β-actin signals. ( B ) The same experiment as in A was performed in MCF7 cells by using a different RFWD3 siRNA (sequence no. 1). ( C ) MCF7 cells were transfected with RFWD3 siRNAs (no. 1 or 2) or a control siRNA. At 48 h posttransfection, cells were irradiated with 2.5 Gy IR. The G 1 checkpoint was measured by the relative S-phase entry at indicated time frames. ( D ) Rescue of the G 1 checkpoint by expressing an RNAi-resistant RFWD3 in U2OS stable shRNA knockdown cells. The cells were transfected with the indicated plasmids and were irradiated with 2.5 Gy IR at 48 hr posttransfection.

    Techniques Used: Transfection, Sequencing, Irradiation, Expressing, shRNA

    RFWD3 stabilizes Mdm2. ( A ) U2OS cells were transfected with RFWD3 siRNAs (no. 1 or 2) or control siRNA. At 48 h posttransfection, cells were irradiated with 10 Gy IR. Quantification of relative Mdm2 levels was normalized by β-actin signals. ( B ) U2OS cells were transfected with control siRNA, RFWD3 siRNA (sequence no. 1), together with empty vector (E.V.) or RNAi-resistant RFWD3. At 48 h posttransfection, cells were irradiated with 10 Gy IR and cell lysates were collected at indicated time points. ( C ) H1299 cells were transfected with E.V. or cotransfected with Mdm2 and various amounts of V5-RFWD3 or V5-RFWD3-CA. GFP was used as an internal control for transfection efficiency. The relative Mdm2 levels were quantified by normalizing to GFP signals. ( D ) MCF7 cells stably transfected with an RFWD3 shRNA (sequence no. 1) or control (scramble) shRNA were treated with cycloheximide for indicated times. ( E ) H1299 cells were transfected with Mdm2 alone or Mdm2 combined with V5-RFWD3 or V5-RFWD3-CA and treated with cycloheximide for indicated times.
    Figure Legend Snippet: RFWD3 stabilizes Mdm2. ( A ) U2OS cells were transfected with RFWD3 siRNAs (no. 1 or 2) or control siRNA. At 48 h posttransfection, cells were irradiated with 10 Gy IR. Quantification of relative Mdm2 levels was normalized by β-actin signals. ( B ) U2OS cells were transfected with control siRNA, RFWD3 siRNA (sequence no. 1), together with empty vector (E.V.) or RNAi-resistant RFWD3. At 48 h posttransfection, cells were irradiated with 10 Gy IR and cell lysates were collected at indicated time points. ( C ) H1299 cells were transfected with E.V. or cotransfected with Mdm2 and various amounts of V5-RFWD3 or V5-RFWD3-CA. GFP was used as an internal control for transfection efficiency. The relative Mdm2 levels were quantified by normalizing to GFP signals. ( D ) MCF7 cells stably transfected with an RFWD3 shRNA (sequence no. 1) or control (scramble) shRNA were treated with cycloheximide for indicated times. ( E ) H1299 cells were transfected with Mdm2 alone or Mdm2 combined with V5-RFWD3 or V5-RFWD3-CA and treated with cycloheximide for indicated times.

    Techniques Used: Transfection, Irradiation, Sequencing, Plasmid Preparation, Stable Transfection, shRNA

    28) Product Images from "Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis"

    Article Title: Genotoxic stress modulates the release of exosomes from multiple myeloma cells capable of activating NK cell cytokine production: Role of HSP70/TLR2/NF-kB axis

    Journal: Oncoimmunology

    doi: 10.1080/2162402X.2017.1279372

    MM exosomes stimulate IFNγ production in a TLR2-dependent manner. (A) NK cells were incubated with increasing doses of Pam3CSK4 or SKO-007(J3) cell-derived exosomes, as indicated. After 24 h, BFA (5 µg/mL) was added and left for additional 24 h. Intracellular IFNγ expression was evaluated by immunofluorescence and FACS analysis. The gating strategy used consists in separating CD56high cells from CD56low NK cells. Numbers indicate the percentage of IFNγ+ cells. A representative experiment is shown. (B) NK cells were incubated with exosomes (20 µg/mL) as described in panel (A). The mean values of four independent experiments ± SEM is shown. (C) NK cells were FACs sorted based on CD56 expression levels, and incubated for 48 h with SKO-007(J3)-derived exosomes (20 µg/mL) or Pam3CSK4 (1 μM). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. The mean values of five independent experiments ± SEM are shown. (D) Cell surface expression of TLR2 was evaluated on CD56 + CD3 − total NK cells, on CD56 high CD3 − and CD56 low CD3 − NK cell subsets of total PBMC derived from 10 different healthy donors. Values represent the mean fluorescence intensity (MFI) of TLR2 substracted from the MFI value of the isotype control Ig. (E) CD56high NK cells were pretreated for 20 min with anti-TLR2 neutralizing antibody (1μg/106 cells), and then incubated with both TLR2 agonist and exosomes as described in panel (B). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. Results relative to two representative donors are shown.
    Figure Legend Snippet: MM exosomes stimulate IFNγ production in a TLR2-dependent manner. (A) NK cells were incubated with increasing doses of Pam3CSK4 or SKO-007(J3) cell-derived exosomes, as indicated. After 24 h, BFA (5 µg/mL) was added and left for additional 24 h. Intracellular IFNγ expression was evaluated by immunofluorescence and FACS analysis. The gating strategy used consists in separating CD56high cells from CD56low NK cells. Numbers indicate the percentage of IFNγ+ cells. A representative experiment is shown. (B) NK cells were incubated with exosomes (20 µg/mL) as described in panel (A). The mean values of four independent experiments ± SEM is shown. (C) NK cells were FACs sorted based on CD56 expression levels, and incubated for 48 h with SKO-007(J3)-derived exosomes (20 µg/mL) or Pam3CSK4 (1 μM). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. The mean values of five independent experiments ± SEM are shown. (D) Cell surface expression of TLR2 was evaluated on CD56 + CD3 − total NK cells, on CD56 high CD3 − and CD56 low CD3 − NK cell subsets of total PBMC derived from 10 different healthy donors. Values represent the mean fluorescence intensity (MFI) of TLR2 substracted from the MFI value of the isotype control Ig. (E) CD56high NK cells were pretreated for 20 min with anti-TLR2 neutralizing antibody (1μg/106 cells), and then incubated with both TLR2 agonist and exosomes as described in panel (B). Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. Results relative to two representative donors are shown.

    Techniques Used: Incubation, Derivative Assay, Expressing, Immunofluorescence, FACS, Real-time Polymerase Chain Reaction, Fluorescence

    HSP70 is associated to MM cell-derived exosomes and contribute to IFNγ production. (A) HSP70 expression on the surface of exosomes was assessed after overnight incubation of exosomes with latex-beads. Exosomes derived from untreated or MEL-treated MM cells were stained with anti-HSP70 mAb or control isotypic Ig. One out of three experiments is shown. (B) Purified NK cells were incubated for 24 h with 20 µg/mL of SKO-007(J3)-derived exosomes in the presence of A8 aptamer or with a scramble peptide both used at 30 μM. Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells or scramble/treated cells considered as calibrators. The mean values of three independent experiments ± SEM are shown. (C–D) Exosomes were extracted from the plasma of PB or BM-derived from MM patients (Pt), lysed in RIPA buffer and ELISA for HSP70 or CD63 was performed. In panel (C), absolute values of both CD63 and HSP70 are shown for five patients. In panel (D), values of HSP70 were normalized with CD63 and used to compare the relative amount of BM HSP70 vs. PB HSP70. Results relative to nine patients are shown.
    Figure Legend Snippet: HSP70 is associated to MM cell-derived exosomes and contribute to IFNγ production. (A) HSP70 expression on the surface of exosomes was assessed after overnight incubation of exosomes with latex-beads. Exosomes derived from untreated or MEL-treated MM cells were stained with anti-HSP70 mAb or control isotypic Ig. One out of three experiments is shown. (B) Purified NK cells were incubated for 24 h with 20 µg/mL of SKO-007(J3)-derived exosomes in the presence of A8 aptamer or with a scramble peptide both used at 30 μM. Real-time PCR analysis of IFNγ mRNA was performed and the data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells or scramble/treated cells considered as calibrators. The mean values of three independent experiments ± SEM are shown. (C–D) Exosomes were extracted from the plasma of PB or BM-derived from MM patients (Pt), lysed in RIPA buffer and ELISA for HSP70 or CD63 was performed. In panel (C), absolute values of both CD63 and HSP70 are shown for five patients. In panel (D), values of HSP70 were normalized with CD63 and used to compare the relative amount of BM HSP70 vs. PB HSP70. Results relative to nine patients are shown.

    Techniques Used: Derivative Assay, Expressing, Incubation, Staining, Purification, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    MM cell-derived exosomes stimulate IFNγ production through a mechanism mediated by NF-kB pathway. (A) NK cells were incubated for 48 h with 20 μg/mL of SKO-007(J3)-derived exosomes. Real-time PCR analysis of IFNγ mRNA. Data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. Values reported represent the mean of six independent experiments ± SEM. (B) NK cells were incubated with 20 μg/mL of SKO-007(J3)-derived exosomes as described in A. Western blot analysis was performed on total cell lysates using p65, phospho-p65 (p-p65) and β-actin Abs. Numbers beneath each line represent quantification of p-p65 and p65 by densitometry normalized with β-actin. (C) NK cells were pretreated for 1 h with the NF-kB inhibitor, SN50 (15 µM), and then incubated with 20 μg/mL of SKO-007(J3)-derived exosomes for 48 h. Real-time PCR analysis of IFNγ mRNA was performed as described in panel (A). The mean of three independent experiments is shown. (D) Nuclear extracts were prepared from NK cells untreated or treated with MM-derived exosomes, and analyzed by EMSA. The nuclear extract derived from NK cells treated with MM exosomes was used for competition with unlabelled probes as indicated in the right panel. (E) NK cells were cultured with 20 μg/mL of SKO-007(J3) cells-derived exosomes in the presence of IL-15 (50 ng/mL). After 24 h, BFA (5 µg/mL) was added and left for additional 24 h. Intracellular IFNγ expression was evaluated by immunofluorescence and FACS analysis. Numbers represent the percentage of IFNγ+ NK cells. One representative experiment is shown. (F) Data were represented as mean values of the percentage of IFNγ+ cells of seven independent experiments ± SEM.
    Figure Legend Snippet: MM cell-derived exosomes stimulate IFNγ production through a mechanism mediated by NF-kB pathway. (A) NK cells were incubated for 48 h with 20 μg/mL of SKO-007(J3)-derived exosomes. Real-time PCR analysis of IFNγ mRNA. Data, expressed as fold change units, were normalized with β-actin and referred to the untreated cells considered as calibrator. Values reported represent the mean of six independent experiments ± SEM. (B) NK cells were incubated with 20 μg/mL of SKO-007(J3)-derived exosomes as described in A. Western blot analysis was performed on total cell lysates using p65, phospho-p65 (p-p65) and β-actin Abs. Numbers beneath each line represent quantification of p-p65 and p65 by densitometry normalized with β-actin. (C) NK cells were pretreated for 1 h with the NF-kB inhibitor, SN50 (15 µM), and then incubated with 20 μg/mL of SKO-007(J3)-derived exosomes for 48 h. Real-time PCR analysis of IFNγ mRNA was performed as described in panel (A). The mean of three independent experiments is shown. (D) Nuclear extracts were prepared from NK cells untreated or treated with MM-derived exosomes, and analyzed by EMSA. The nuclear extract derived from NK cells treated with MM exosomes was used for competition with unlabelled probes as indicated in the right panel. (E) NK cells were cultured with 20 μg/mL of SKO-007(J3) cells-derived exosomes in the presence of IL-15 (50 ng/mL). After 24 h, BFA (5 µg/mL) was added and left for additional 24 h. Intracellular IFNγ expression was evaluated by immunofluorescence and FACS analysis. Numbers represent the percentage of IFNγ+ NK cells. One representative experiment is shown. (F) Data were represented as mean values of the percentage of IFNγ+ cells of seven independent experiments ± SEM.

    Techniques Used: Derivative Assay, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Cell Culture, Expressing, Immunofluorescence, FACS

    29) Product Images from "Genetic deficiency of Syk protects mice from autoantibody-induced arthritis"

    Article Title: Genetic deficiency of Syk protects mice from autoantibody-induced arthritis

    Journal: Arthritis and Rheumatism

    doi: 10.1002/art.27438

    Generation and analysis of Syk −/− bone marrow chimeric mice. A , Macroscopic appearance of wild-type (WT) and Syk −/− mouse fetuses at ∼17.5 days postcoitum (embryogenesis day 17.5). B , General scheme of fetal liver transplantation procedure. C , Flow cytometric analysis of donor marker (CD45.2) expression in all circulating leukocytes (CD45+ gate), circulating neutrophils (Gr-1+ gate), circulating monocytes (CD11b+Gr-1– gate), and bone marrow (BM)–derived macrophages (F4/80+ gate) from intact (nonchimeric) mice of the CD45.1-expressing recipient strain or the CD45.2-expressing donor (C57BL/6) strain, as well as from wild-type or Syk −/− bone marrow chimeras. D , Immunoblot analysis of Syk and β-actin protein levels in total bone marrow cells or bone marrow–derived macrophages. Results are representative of 3 or more independent experiments, each of which showed similar results. Actin was used as a loading control.
    Figure Legend Snippet: Generation and analysis of Syk −/− bone marrow chimeric mice. A , Macroscopic appearance of wild-type (WT) and Syk −/− mouse fetuses at ∼17.5 days postcoitum (embryogenesis day 17.5). B , General scheme of fetal liver transplantation procedure. C , Flow cytometric analysis of donor marker (CD45.2) expression in all circulating leukocytes (CD45+ gate), circulating neutrophils (Gr-1+ gate), circulating monocytes (CD11b+Gr-1– gate), and bone marrow (BM)–derived macrophages (F4/80+ gate) from intact (nonchimeric) mice of the CD45.1-expressing recipient strain or the CD45.2-expressing donor (C57BL/6) strain, as well as from wild-type or Syk −/− bone marrow chimeras. D , Immunoblot analysis of Syk and β-actin protein levels in total bone marrow cells or bone marrow–derived macrophages. Results are representative of 3 or more independent experiments, each of which showed similar results. Actin was used as a loading control.

    Techniques Used: Mouse Assay, Transplantation Assay, Flow Cytometry, Marker, Expressing, Derivative Assay

    30) Product Images from "Direct Pro-Inflammatory Effects of Prorenin on Microglia"

    Article Title: Direct Pro-Inflammatory Effects of Prorenin on Microglia

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0092937

    PRR expression and function in microglia. ( A ) (i) Representative fluorescence micrographs showing immunoreactive PRR (P) and Iba1 co-localized (P+I) in mouse N-9 microglial cells and SD rat primary microglia. ( B ) CD11b protein expression was analyzed in N-9 cells and SD rat primary microglia by western blotting following 24 hr treatment with either control media (DMEM) or prorenin (20 nmol/L). Top : Representative immunoblots showing CD11b and β-actin (loading control) protein bands under each treatment condition; Bottom : Bar graphs are band density ratios of CD11b protein normalized against β-actin. Data are means ± SEM, n = 6 experiments.
    Figure Legend Snippet: PRR expression and function in microglia. ( A ) (i) Representative fluorescence micrographs showing immunoreactive PRR (P) and Iba1 co-localized (P+I) in mouse N-9 microglial cells and SD rat primary microglia. ( B ) CD11b protein expression was analyzed in N-9 cells and SD rat primary microglia by western blotting following 24 hr treatment with either control media (DMEM) or prorenin (20 nmol/L). Top : Representative immunoblots showing CD11b and β-actin (loading control) protein bands under each treatment condition; Bottom : Bar graphs are band density ratios of CD11b protein normalized against β-actin. Data are means ± SEM, n = 6 experiments.

    Techniques Used: Expressing, Fluorescence, Western Blot

    31) Product Images from "Increasing SK channel activity reverses ischemia-induced impairment of LTP"

    Article Title: Increasing SK channel activity reverses ischemia-induced impairment of LTP

    Journal: The European journal of neuroscience

    doi: 10.1111/ejn.12683

    Ischemia does not alter AMPA/NMDA ratio. A ) Representative EPSCs from sham control. Application of NBQX (red trace) inhibits the AMPA component of the initial EPSC (black trace). Subsequent application of D-APV inhibits to remaining NMDA component (blue trace). B ) Quantification of AMPA to NMDA ratio for all cells recorded demonstrates no difference of CA/CPR. C ) Quantification of mRNA expression of the 3 predominant isoforms of NMDA receptor expressed in the hippocampus, GluN1, 2A and 2B. D ) Representative Western blot analysis of NR1, PSD95 and β-actin in synaptic membrane preparations from hippocampi obtained at either 7 or 30 days after CA/CPR, or sham control mice. Quantification (bottom) of NR1:PSD95 ratio shows not changes in response to CA/CPR. Data presented as mean ± SEM.
    Figure Legend Snippet: Ischemia does not alter AMPA/NMDA ratio. A ) Representative EPSCs from sham control. Application of NBQX (red trace) inhibits the AMPA component of the initial EPSC (black trace). Subsequent application of D-APV inhibits to remaining NMDA component (blue trace). B ) Quantification of AMPA to NMDA ratio for all cells recorded demonstrates no difference of CA/CPR. C ) Quantification of mRNA expression of the 3 predominant isoforms of NMDA receptor expressed in the hippocampus, GluN1, 2A and 2B. D ) Representative Western blot analysis of NR1, PSD95 and β-actin in synaptic membrane preparations from hippocampi obtained at either 7 or 30 days after CA/CPR, or sham control mice. Quantification (bottom) of NR1:PSD95 ratio shows not changes in response to CA/CPR. Data presented as mean ± SEM.

    Techniques Used: Expressing, Western Blot, Mouse Assay

    32) Product Images from "Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma"

    Article Title: Oncogenic activation of the RNA binding protein NELFE and MYC signaling in hepatocellular carcinoma

    Journal: Cancer cell

    doi: 10.1016/j.ccell.2017.06.002

    NELFE enhances MYC tumorigenicity. (A) Bar graph of colony formation assay of HHT4 cells or HHT4 cells ectopically expressing the indicated proteins at day 10. (B) Representatie image and quantifiation of oncosphere formation assay at day 7 measured by Algimatrix 3D assay. Scale bar, 200 μM. (C) Proliferation rates of different cell lines up to 72 hr. (D) RT-PCR analysis of relative mRNA expression of MYC-related genes. (E) Hematoxylin and eosin and immunohistochemical staining of indicated tumors. Scale bars, 40 μM. (F) Number of tumor nodules four weeks aftr the injection of indicated cells. Short horizontal lines represent the mean. (G) RT-PCR analysis of relative mRNA expression of MYC-related genes in MYC or MYC+NELFE tumor tissues. Data were first normalized to β–actin to get dCt. Relative mRNA was then calculated using 2 dCt .
    Figure Legend Snippet: NELFE enhances MYC tumorigenicity. (A) Bar graph of colony formation assay of HHT4 cells or HHT4 cells ectopically expressing the indicated proteins at day 10. (B) Representatie image and quantifiation of oncosphere formation assay at day 7 measured by Algimatrix 3D assay. Scale bar, 200 μM. (C) Proliferation rates of different cell lines up to 72 hr. (D) RT-PCR analysis of relative mRNA expression of MYC-related genes. (E) Hematoxylin and eosin and immunohistochemical staining of indicated tumors. Scale bars, 40 μM. (F) Number of tumor nodules four weeks aftr the injection of indicated cells. Short horizontal lines represent the mean. (G) RT-PCR analysis of relative mRNA expression of MYC-related genes in MYC or MYC+NELFE tumor tissues. Data were first normalized to β–actin to get dCt. Relative mRNA was then calculated using 2 dCt .

    Techniques Used: Colony Assay, Expressing, Tube Formation Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemistry, Staining, Injection

    33) Product Images from "Cbl-b Is a Critical Regulator of Macrophage Activation Associated With Obesity-Induced Insulin Resistance in Mice"

    Article Title: Cbl-b Is a Critical Regulator of Macrophage Activation Associated With Obesity-Induced Insulin Resistance in Mice

    Journal: Diabetes

    doi: 10.2337/db12-0677

    Role of Cbl-b in TLR4-mediated saturated FA signaling. A : HEK293/TLR4 cells were transfected with TLR4-V5, MyD88-Myc, mock vector/pCEFL-Cbl-b-HA, and pcDNA3-FLAG-Ubiquitin. To analyze the degradation rates of saturated FA signaling molecules, these cells were treated with 100 μg/mL cycloheximide. After treatment with palmitate for the indicated time intervals, whole-cell lysates were subjected to immunoblotting (IB; 20 μg/lane) for the indicated proteins. Each experiment was independently reproduced three times. Data are densitometric ratio of V5 to β-actin and are mean ± SD values of three experiments. * P
    Figure Legend Snippet: Role of Cbl-b in TLR4-mediated saturated FA signaling. A : HEK293/TLR4 cells were transfected with TLR4-V5, MyD88-Myc, mock vector/pCEFL-Cbl-b-HA, and pcDNA3-FLAG-Ubiquitin. To analyze the degradation rates of saturated FA signaling molecules, these cells were treated with 100 μg/mL cycloheximide. After treatment with palmitate for the indicated time intervals, whole-cell lysates were subjected to immunoblotting (IB; 20 μg/lane) for the indicated proteins. Each experiment was independently reproduced three times. Data are densitometric ratio of V5 to β-actin and are mean ± SD values of three experiments. * P

    Techniques Used: Transfection, Plasmid Preparation

    34) Product Images from "Binding of LBP-1a to Specific Immunoglobulin Switch Regions in vivo Correlates with Specific Repression of Class Switch Recombination"

    Article Title: Binding of LBP-1a to Specific Immunoglobulin Switch Regions in vivo Correlates with Specific Repression of Class Switch Recombination

    Journal: European journal of immunology

    doi: 10.1002/eji.200838226

    LBP-1a binds to Sμ and Sα, but not Sγ1, regions in resting B cells and is released after LPS stimulation. A) Schematic of mouse immunoglobulin S region sequences with predicted LSF/LBP-1a binding sites. Long horizontal lines, to scale, represent each S region sequence; intersecting vertical lines represent predicted LSF/LBP-1a binding sites. Shorter horizontal lines below each S region represent regions amplified in ChIP assays. Amplicon sequences are in boxes below each S region schematic; PCR primers are in bold. Predicted LSF/LBP-1a binding sites within or flanking each amplicon are in shaded boxes. Brackets indicate overlapping sites. B) Isolated primary splenic murine B-lymphocytes were analyzed by ChIP with LBP-1a antiserum. Negative controls included non-specific rabbit IgG (IgG) and preimmune rabbit serum (PI). Input represents 1.25% of ChIP starting material. DNA were amplified with the primers in (A). Data are representative of at least 3 independent experiments. C) Whole cell extracts from 10 × 10 6 B-cells, stimulated with 50 µg/ml LPS for the indicated amounts of time, were immunoblotted (IB) with antibodies against LBP-1a or β-actin, as indicated. D–E) Resting B-cells were stimulated with 50 µg/ml LPS for the indicated times; LBP-1a occupancy at Sμ (D) and Sα (E) were analyzed as in (B). Upper panel: ChIP analysis with nonspecific rabbit IgG (−) or LBP-1a antiserum (+) at the indicated hours post-LPS stimulation. Lower panel: Amplication of input samples for each time point representing 0.125% (odd lanes) or 0.0125% (even lanes) of the ChIP starting material. Data are representative of 4 independent experiments.
    Figure Legend Snippet: LBP-1a binds to Sμ and Sα, but not Sγ1, regions in resting B cells and is released after LPS stimulation. A) Schematic of mouse immunoglobulin S region sequences with predicted LSF/LBP-1a binding sites. Long horizontal lines, to scale, represent each S region sequence; intersecting vertical lines represent predicted LSF/LBP-1a binding sites. Shorter horizontal lines below each S region represent regions amplified in ChIP assays. Amplicon sequences are in boxes below each S region schematic; PCR primers are in bold. Predicted LSF/LBP-1a binding sites within or flanking each amplicon are in shaded boxes. Brackets indicate overlapping sites. B) Isolated primary splenic murine B-lymphocytes were analyzed by ChIP with LBP-1a antiserum. Negative controls included non-specific rabbit IgG (IgG) and preimmune rabbit serum (PI). Input represents 1.25% of ChIP starting material. DNA were amplified with the primers in (A). Data are representative of at least 3 independent experiments. C) Whole cell extracts from 10 × 10 6 B-cells, stimulated with 50 µg/ml LPS for the indicated amounts of time, were immunoblotted (IB) with antibodies against LBP-1a or β-actin, as indicated. D–E) Resting B-cells were stimulated with 50 µg/ml LPS for the indicated times; LBP-1a occupancy at Sμ (D) and Sα (E) were analyzed as in (B). Upper panel: ChIP analysis with nonspecific rabbit IgG (−) or LBP-1a antiserum (+) at the indicated hours post-LPS stimulation. Lower panel: Amplication of input samples for each time point representing 0.125% (odd lanes) or 0.0125% (even lanes) of the ChIP starting material. Data are representative of 4 independent experiments.

    Techniques Used: Binding Assay, Sequencing, Amplification, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Isolation

    LBP-1a is the predominant LSF family member in primary B cells. A) Sequences of LBP-1a (aa 274–336) and LSF (aa 277–332) are aligned. Antipeptide antibodies were produced against nonconserved epitopes (bold). B) Ten ng of His-LBP-1a or His-LSF were subjected to SDS-PAGE and immunoblotted (IB) with the anti-peptide antibodies specific to LBP-1a or LSF, as indicated. C) Extracts (50 µg) from untransfected 293T cells, cells transfected with pCxLSF, or cells transfected with pCxLBP-1a were separated by SDS-PAGE through a 7.5% gel and immunoblotted (IB) with the antipeptide antibodies specific to LSF or LBP-1a, as indicated. D) Left panels: Decreasing levels of purified His-LSF or His-LBP-1a were immunoblotted with the respective antipeptide antibodies. Right panels: on the same gels, whole cell lysates from 20 × 10 6 splenic mouse B-cells in a single experiment were immunoblotted with antipeptide antibodies specific to either LSF (upper panels) or LBP-1a (lower panels), and subsequently with antibody against β-actin.
    Figure Legend Snippet: LBP-1a is the predominant LSF family member in primary B cells. A) Sequences of LBP-1a (aa 274–336) and LSF (aa 277–332) are aligned. Antipeptide antibodies were produced against nonconserved epitopes (bold). B) Ten ng of His-LBP-1a or His-LSF were subjected to SDS-PAGE and immunoblotted (IB) with the anti-peptide antibodies specific to LBP-1a or LSF, as indicated. C) Extracts (50 µg) from untransfected 293T cells, cells transfected with pCxLSF, or cells transfected with pCxLBP-1a were separated by SDS-PAGE through a 7.5% gel and immunoblotted (IB) with the antipeptide antibodies specific to LSF or LBP-1a, as indicated. D) Left panels: Decreasing levels of purified His-LSF or His-LBP-1a were immunoblotted with the respective antipeptide antibodies. Right panels: on the same gels, whole cell lysates from 20 × 10 6 splenic mouse B-cells in a single experiment were immunoblotted with antipeptide antibodies specific to either LSF (upper panels) or LBP-1a (lower panels), and subsequently with antibody against β-actin.

    Techniques Used: Produced, SDS Page, Transfection, Purification

    35) Product Images from "14-3-3? is a Binding Partner of Rat Eag1 Potassium Channels"

    Article Title: 14-3-3? is a Binding Partner of Rat Eag1 Potassium Channels

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041203

    Lack of effect of 14-3-3θ over-expression on the total and surface expression of rEag1 protein. ( A ) Representative result of surface biotinylation experiments. Intact HEK293T cells were biontylinated on ice and thereafter solubilized. ( Surface ) Cell lysates were pulled down with streptavidin agarose beads, followed by immunoblotting with the anti-rEag1 antibody. ( Input ) Cell lysates were directly employed for immunoblotting analyses. Input represents 5% of the total protein used for streptavidin pull-down. Also shown at the bottom are the corresponding β-actin expression levels for each lane. The specificity of the biotinylation procedure was verified by the absence of β-actin bands in the surface fraction. ( B ) Quantification of total and surface expression of rEag1 in the absence or presence of 14-3-3θ over-expression. The total protein density ( top panel ) was determined as the ratio of input signal to the cognate β-actin signal. The surface expression efficiency ( bottom panel ) was expressed as the ratio of surface signal to the corresponding total protein density. The mean values were subsequently normalized with respect to that of vector control. Densitometric scans of immunoblots were obtained from three independent experiments.
    Figure Legend Snippet: Lack of effect of 14-3-3θ over-expression on the total and surface expression of rEag1 protein. ( A ) Representative result of surface biotinylation experiments. Intact HEK293T cells were biontylinated on ice and thereafter solubilized. ( Surface ) Cell lysates were pulled down with streptavidin agarose beads, followed by immunoblotting with the anti-rEag1 antibody. ( Input ) Cell lysates were directly employed for immunoblotting analyses. Input represents 5% of the total protein used for streptavidin pull-down. Also shown at the bottom are the corresponding β-actin expression levels for each lane. The specificity of the biotinylation procedure was verified by the absence of β-actin bands in the surface fraction. ( B ) Quantification of total and surface expression of rEag1 in the absence or presence of 14-3-3θ over-expression. The total protein density ( top panel ) was determined as the ratio of input signal to the cognate β-actin signal. The surface expression efficiency ( bottom panel ) was expressed as the ratio of surface signal to the corresponding total protein density. The mean values were subsequently normalized with respect to that of vector control. Densitometric scans of immunoblots were obtained from three independent experiments.

    Techniques Used: Over Expression, Expressing, Plasmid Preparation, Western Blot

    Phosphorylation-independent interaction of rEag1 with 14-3-3θ. ( A ) Co-immunoprecipitation of myc-14-3-3θ and rEag1 proteins. ( Left panel ) rEag1/rEag2 was co-expressed with an empty vector ( − ) or myc-tagged 14-3-3θ ( + ) in HEK293T cells. Cell lysates were immunoprecipitated ( IP ) by using the anti-myc antibody, followed by immunoblotting ( WB ) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3θ are highlighted with arrow and arrowhead, respectively. ( Right panel ) Cell lysates from myc-14-3-3θ only or co-expression of rEag1 and myc-14-3-3θ were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. ( B ) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3θ in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 µM okadaic acid or staurosporine. ( Upper panel ) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. β-actin was run as a loading control. ( Lower panel ) Cell lysates were immunoprecipitated ( IP ) by using the anti-myc antibody, followed by immunoblotting ( WB ) with the anti-myc or the anti-rEag1 antibody. ( C ) Quantification of ( upper panel ) the Akt phosphorylation level ( pAkt/Akt ) and ( lower panel ) the co-immunoprecipitation ( CO-IP ) efficiency of 14-3-3θ and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. The mean values were subsequently normalized with respect to that of the no-treatment control of 14-3-3θ/rEag1 co-expression. Densitometric scans of immunoblots were obtained from three independent experiments. Asterisk denotes a significant difference from the no-treatment control of 14-3-3θ/rEag1 co-expression (*, t -test: p
    Figure Legend Snippet: Phosphorylation-independent interaction of rEag1 with 14-3-3θ. ( A ) Co-immunoprecipitation of myc-14-3-3θ and rEag1 proteins. ( Left panel ) rEag1/rEag2 was co-expressed with an empty vector ( − ) or myc-tagged 14-3-3θ ( + ) in HEK293T cells. Cell lysates were immunoprecipitated ( IP ) by using the anti-myc antibody, followed by immunoblotting ( WB ) with the anti-myc or the anti-rEag1/rEag2 antibody. The protein bands corresponding to rEag1/rEag2 and 14-3-3θ are highlighted with arrow and arrowhead, respectively. ( Right panel ) Cell lysates from myc-14-3-3θ only or co-expression of rEag1 and myc-14-3-3θ were immunoprecipitated by using the anti-rEag1 antibody. Input volumes correspond to 5% of the total cell lysates used for immunoprecipitation. These co-immunoprecipitation data are representative of three to five independent experiments. ( B ) rEag1 was co-expressed with an empty vector or myc-tagged 14-3-3θ in HEK293T cells. 24 hrs after transfection, indicated cells were subject to 1-hr treatment with 1 µM okadaic acid or staurosporine. ( Upper panel ) Total cell lysates were immunoblotted with the anti-Akt (total Akt) or anti-phosphorylated Akt (pAkt) antibodies to monitor the cellular phosphorylation status. β-actin was run as a loading control. ( Lower panel ) Cell lysates were immunoprecipitated ( IP ) by using the anti-myc antibody, followed by immunoblotting ( WB ) with the anti-myc or the anti-rEag1 antibody. ( C ) Quantification of ( upper panel ) the Akt phosphorylation level ( pAkt/Akt ) and ( lower panel ) the co-immunoprecipitation ( CO-IP ) efficiency of 14-3-3θ and rEag1. The CO-IP efficiency was determined by the ratio of the protein band intensities of immunoprecipitated rEag1 to those of cognate total inputs. The mean values were subsequently normalized with respect to that of the no-treatment control of 14-3-3θ/rEag1 co-expression. Densitometric scans of immunoblots were obtained from three independent experiments. Asterisk denotes a significant difference from the no-treatment control of 14-3-3θ/rEag1 co-expression (*, t -test: p

    Techniques Used: Immunoprecipitation, Plasmid Preparation, Western Blot, Expressing, Transfection, Co-Immunoprecipitation Assay

    36) Product Images from "Arginine methylation of SKN-1 promotes oxidative stress resistance in Caenorhabditis elegans"

    Article Title: Arginine methylation of SKN-1 promotes oxidative stress resistance in Caenorhabditis elegans

    Journal: Redox Biology

    doi: 10.1016/j.redox.2019.101111

    Oxidative stress enhances binding of PRMT-1 to SKN-1, and elevates asymmetrical dimethylation of arginines on SKN-1. (A) GST-PRMT-1 bound to SKN-1 proteins expressed in wild-type worms. GST pull-down assay was performed by immunoprecipitation with GST-PRMT-1 protein from the whole extracts of N2[SKN-1B/C::GFP] worms, followed by immunoblotting with anti-GFP antibody. The arrow on the right indicated the band of GST-PRMT-1 purifed from E. coli BL21. GST protein was as the negative control. Coomassie brilliant blue (CBB) staining (lower) was used as a loading control. (B) SKN-1 interacted with PRMT-1 and was asymmetrically dimethylated on arginines in vivo . co-IP was operated by utilizing the whole extracts of N2[SKN-1B/C::GFP] worms with anti-GFP antibody, followed by immunoblotting with anti-PRMT-1 and anti-ASYM antibody. No antibody was used as the negative control. (C) Oxidative stress increased the binding of PRMT-1 to SKN-1, and elevated asymmetrical dimethylation of arginines on SKN-1. N2[SKN-1B/C::GFP] worms were treated with (or without) 5 mM tBHP, followed by co-IP as the legend to B. β-actin and GFP were utilized as internal references. IgG was used as the negative control.
    Figure Legend Snippet: Oxidative stress enhances binding of PRMT-1 to SKN-1, and elevates asymmetrical dimethylation of arginines on SKN-1. (A) GST-PRMT-1 bound to SKN-1 proteins expressed in wild-type worms. GST pull-down assay was performed by immunoprecipitation with GST-PRMT-1 protein from the whole extracts of N2[SKN-1B/C::GFP] worms, followed by immunoblotting with anti-GFP antibody. The arrow on the right indicated the band of GST-PRMT-1 purifed from E. coli BL21. GST protein was as the negative control. Coomassie brilliant blue (CBB) staining (lower) was used as a loading control. (B) SKN-1 interacted with PRMT-1 and was asymmetrically dimethylated on arginines in vivo . co-IP was operated by utilizing the whole extracts of N2[SKN-1B/C::GFP] worms with anti-GFP antibody, followed by immunoblotting with anti-PRMT-1 and anti-ASYM antibody. No antibody was used as the negative control. (C) Oxidative stress increased the binding of PRMT-1 to SKN-1, and elevated asymmetrical dimethylation of arginines on SKN-1. N2[SKN-1B/C::GFP] worms were treated with (or without) 5 mM tBHP, followed by co-IP as the legend to B. β-actin and GFP were utilized as internal references. IgG was used as the negative control.

    Techniques Used: Binding Assay, Pull Down Assay, Immunoprecipitation, Negative Control, Staining, In Vivo, Co-Immunoprecipitation Assay

    37) Product Images from "Plasma Lactoferrin Levels Positively Correlate with Insulin Resistance despite an Inverse Association with Total Adiposity in Lean and Severely Obese Patients"

    Article Title: Plasma Lactoferrin Levels Positively Correlate with Insulin Resistance despite an Inverse Association with Total Adiposity in Lean and Severely Obese Patients

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166138

    Intestinal Lf mRNA and protein expression in severely obese subjects and in Caco-2/15 cells. mRNA and protein levels of Lf were estimated in the intestine of insulin-sensitive and insulin-resistant obese subjects (n = 9 per group). The relative mRNA fold-changes between groups were calculated using the 2 −ΔΔCt method. mRNA data were normalized to ATP5O mRNA expression. Modulation of Lf protein following a 24-hour Caco-2/15 cell incubation with LPS (150 μg/mL). Protein expression values were normalized to β-actin protein expression. In B, samples were run on the same gel, but lanes were not contiguous. * P
    Figure Legend Snippet: Intestinal Lf mRNA and protein expression in severely obese subjects and in Caco-2/15 cells. mRNA and protein levels of Lf were estimated in the intestine of insulin-sensitive and insulin-resistant obese subjects (n = 9 per group). The relative mRNA fold-changes between groups were calculated using the 2 −ΔΔCt method. mRNA data were normalized to ATP5O mRNA expression. Modulation of Lf protein following a 24-hour Caco-2/15 cell incubation with LPS (150 μg/mL). Protein expression values were normalized to β-actin protein expression. In B, samples were run on the same gel, but lanes were not contiguous. * P

    Techniques Used: Expressing, Incubation

    38) Product Images from "Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2"

    Article Title: Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2

    Journal: Nature Communications

    doi: 10.1038/ncomms15728

    Senescence induces increased secretion of sEV-associated EphA2. ( a ) Comparative proteomic analysis of sEVs secreted from control and DXR-induced senescent RPE-1 cells by using mass spectrometry. The number of peptides detected in sEVs secreted from control and DXR-induced senescent RPE-1 cells are plotted for the identified proteins. Blue and red plots represent the data for the proteins significantly enriched in sEVs secreted from control and DXR-induced senescent RPE-1 cells, respectively. Green plots represent the data for the other proteins. ( b ) Immunoblotting of EphA2, Alix and β-actin in the sEV fraction and WCL of control and DXR-induced senescent RPE-1 cells. The same number of sEVs was loaded in each lane. Dot plot represents the relative density of sEV-associated EphA2 analysed by ImageJ. ( c ) Immunoblotting of indicated proteins in the sEV fraction, CM and WCL of DXR-induced senescent RPE-1 cells expressing non-targeting shRNA (shNT) or Rab35 shRNA (shRab35). Dot plot represents the relative density of sEV-associated EphA2 analysed by ImageJ. ( d ) Immunoblotting of indicated proteins in the sEV fraction and WCL of pre-senescent control and senescent TIG-3 cells. Senescence was induced by serial passage, oncogenic Ras expression or DXR-treatment. The same number of sEVs was loaded in each lane. Dot plots represent the relative density of sEV-associated EphA2 analysed by ImageJ. Replicates are biological replicates ( n =3). Error bars indicate s.d. * P
    Figure Legend Snippet: Senescence induces increased secretion of sEV-associated EphA2. ( a ) Comparative proteomic analysis of sEVs secreted from control and DXR-induced senescent RPE-1 cells by using mass spectrometry. The number of peptides detected in sEVs secreted from control and DXR-induced senescent RPE-1 cells are plotted for the identified proteins. Blue and red plots represent the data for the proteins significantly enriched in sEVs secreted from control and DXR-induced senescent RPE-1 cells, respectively. Green plots represent the data for the other proteins. ( b ) Immunoblotting of EphA2, Alix and β-actin in the sEV fraction and WCL of control and DXR-induced senescent RPE-1 cells. The same number of sEVs was loaded in each lane. Dot plot represents the relative density of sEV-associated EphA2 analysed by ImageJ. ( c ) Immunoblotting of indicated proteins in the sEV fraction, CM and WCL of DXR-induced senescent RPE-1 cells expressing non-targeting shRNA (shNT) or Rab35 shRNA (shRab35). Dot plot represents the relative density of sEV-associated EphA2 analysed by ImageJ. ( d ) Immunoblotting of indicated proteins in the sEV fraction and WCL of pre-senescent control and senescent TIG-3 cells. Senescence was induced by serial passage, oncogenic Ras expression or DXR-treatment. The same number of sEVs was loaded in each lane. Dot plots represent the relative density of sEV-associated EphA2 analysed by ImageJ. Replicates are biological replicates ( n =3). Error bars indicate s.d. * P

    Techniques Used: Mass Spectrometry, Expressing, shRNA

    Ephrin-A1 mediates the growth-promoting effect of sEV-associated EphA2. ( a ) Immunoblotting of EphA2, ephrin-A1 and β-actin in the WCL of indicated cell lines. ( b ) Relative numbers of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. Normal rabbit IgG or rabbit anti-ephrin-A1 IgG (AER-031; Alomone) was added to the medium at a concentration of 5 μg ml −1 . CM was prepared from DXR-induced senescent RPE-1 cells. ( c ) Immunoblotting shows successful overexpression of ephrin-A1 in MCF-7 cells. Dot plot represents the relative numbers of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. Empty vector or ectopic ephrin-A1 was expressed in MCF-7 cells. CM was prepared from DXR-induced senescent RPE-1 cells. Replicates are biological replicates ( n =3 for b and n =5 for c ). Error bars indicate s.d. * P
    Figure Legend Snippet: Ephrin-A1 mediates the growth-promoting effect of sEV-associated EphA2. ( a ) Immunoblotting of EphA2, ephrin-A1 and β-actin in the WCL of indicated cell lines. ( b ) Relative numbers of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. Normal rabbit IgG or rabbit anti-ephrin-A1 IgG (AER-031; Alomone) was added to the medium at a concentration of 5 μg ml −1 . CM was prepared from DXR-induced senescent RPE-1 cells. ( c ) Immunoblotting shows successful overexpression of ephrin-A1 in MCF-7 cells. Dot plot represents the relative numbers of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. Empty vector or ectopic ephrin-A1 was expressed in MCF-7 cells. CM was prepared from DXR-induced senescent RPE-1 cells. Replicates are biological replicates ( n =3 for b and n =5 for c ). Error bars indicate s.d. * P

    Techniques Used: Concentration Assay, Over Expression, Plasmid Preparation

    sEV-associated EphA2 promotes cancer cell proliferation through EphA2/ephrin-A1 reverse signalling. ( a ) Immunoblotting of EphA2 and β-actin in the WCL of MCF-7 cells expressing empty vector or ectopic EphA2. Cells were grown for 3 days with or without CM prepared from DXR-induced senescent RPE-1 cells. ( b ) EphA2 immunoprecipitates prepared from MCF-7 cells expressing ectopic EphA2 were immunoblotted with anti-phosphotyrosine and anti-EphA2 antibody. The indicated samples were treated with recombinant human ephrin-A1 (500 ng ml −1 ) for 20 min. Eight-hour pre-treatment with the EphA2 inhibitor ALW-II-41-27 (100 nM) suppressed ephrin-A1-induced EphA2 phosphorylation. ( c ) Relative numbers of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. DMSO or ALW-II-41-27 (100 nM) was added to the medium. ( d ) Immunoblotting of phospho-Erk, total-Erk and β-actin in the WCL of MCF-7 cells grown for 3 days with or without CM prepared from control or DXR-induced senescent RPE-1 cells expressing non-targeting shRNA (shNT), shEphA2 or shRab35. ( e ) The heatmap shows the relative gene expression levels in MCF-7 cells grown for 3 days with or without CM prepared from DXR-induced senescent RPE-1 cells expressing shNT or shEphA2. Genes whose expression levels were changed more than 1.2-fold by the CM of shNT-expressing cells are shown. ( f ) EphA2-dependent upregulated genes were analysed with oPOSSUM-3. Each circle in the plot shows an enrichment of the targets of a different transcription factor. ( g ) Immunoblotting of phospho-Erk, total-Erk and β-actin in the WCL of MCF-7 cells grown for 3 days with or without CM. Dot plot represents the number of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. DMSO or U0126 (500 nM) was added to the medium. CM was prepared from DXR-induced senescent RPE-1 cells. Replicates are biological replicates ( n =3). Error bars indicate s.d. * P
    Figure Legend Snippet: sEV-associated EphA2 promotes cancer cell proliferation through EphA2/ephrin-A1 reverse signalling. ( a ) Immunoblotting of EphA2 and β-actin in the WCL of MCF-7 cells expressing empty vector or ectopic EphA2. Cells were grown for 3 days with or without CM prepared from DXR-induced senescent RPE-1 cells. ( b ) EphA2 immunoprecipitates prepared from MCF-7 cells expressing ectopic EphA2 were immunoblotted with anti-phosphotyrosine and anti-EphA2 antibody. The indicated samples were treated with recombinant human ephrin-A1 (500 ng ml −1 ) for 20 min. Eight-hour pre-treatment with the EphA2 inhibitor ALW-II-41-27 (100 nM) suppressed ephrin-A1-induced EphA2 phosphorylation. ( c ) Relative numbers of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. DMSO or ALW-II-41-27 (100 nM) was added to the medium. ( d ) Immunoblotting of phospho-Erk, total-Erk and β-actin in the WCL of MCF-7 cells grown for 3 days with or without CM prepared from control or DXR-induced senescent RPE-1 cells expressing non-targeting shRNA (shNT), shEphA2 or shRab35. ( e ) The heatmap shows the relative gene expression levels in MCF-7 cells grown for 3 days with or without CM prepared from DXR-induced senescent RPE-1 cells expressing shNT or shEphA2. Genes whose expression levels were changed more than 1.2-fold by the CM of shNT-expressing cells are shown. ( f ) EphA2-dependent upregulated genes were analysed with oPOSSUM-3. Each circle in the plot shows an enrichment of the targets of a different transcription factor. ( g ) Immunoblotting of phospho-Erk, total-Erk and β-actin in the WCL of MCF-7 cells grown for 3 days with or without CM. Dot plot represents the number of MCF-7 cells grown for 3 days in the presence of CM compared with the number of cells grown for 3 days in normal medium. DMSO or U0126 (500 nM) was added to the medium. CM was prepared from DXR-induced senescent RPE-1 cells. Replicates are biological replicates ( n =3). Error bars indicate s.d. * P

    Techniques Used: Expressing, Plasmid Preparation, Recombinant, shRNA

    Senescence increases the secretion of sEVs. Immunoblotting of Alix (exosome marker) and CD9 (sEV marker) in the sEV fraction and of phospho-p53 (Ser15), p21 and β-actin in the whole cell lysates (WCL) of pre-senescent control and senescent cells. Senescence was induced by serial passage, oncogenic Ras expression or DXR-treatment in TIG-3 or RPE-1 cells. Dot plots show the relative numbers of sEVs per cell. The number of sEVs in the sEV fraction was quantified using NanoSight. The bottom graphs show the size distributions of sEVs secreted from control and senescent cells. Cell densities were as following at 1 day before starting CM preparation for sEV purification; subconfluent for pre-senescent TIG-3 and RPE-1 cells; 8 × 10 2 cells cm −2 for replicative senescent TIG-3 cells; 1 × 10 4 cells cm −2 for Ras-induced senescent TIG-3 cells; 5 × 10 3 cells cm −2 for DXR-induced senescent TIG-3 cells; 1.4 × 10 4 cells cm −2 for DXR-induced senescent RPE-1 cells. The CM to be used for sEV purification was prepared by culturing the cells in DMEM supplemented 5% EV-depleted FBS for 3 days. Replicates are biological replicates ( n =3). Error bars indicate s.d. * P
    Figure Legend Snippet: Senescence increases the secretion of sEVs. Immunoblotting of Alix (exosome marker) and CD9 (sEV marker) in the sEV fraction and of phospho-p53 (Ser15), p21 and β-actin in the whole cell lysates (WCL) of pre-senescent control and senescent cells. Senescence was induced by serial passage, oncogenic Ras expression or DXR-treatment in TIG-3 or RPE-1 cells. Dot plots show the relative numbers of sEVs per cell. The number of sEVs in the sEV fraction was quantified using NanoSight. The bottom graphs show the size distributions of sEVs secreted from control and senescent cells. Cell densities were as following at 1 day before starting CM preparation for sEV purification; subconfluent for pre-senescent TIG-3 and RPE-1 cells; 8 × 10 2 cells cm −2 for replicative senescent TIG-3 cells; 1 × 10 4 cells cm −2 for Ras-induced senescent TIG-3 cells; 5 × 10 3 cells cm −2 for DXR-induced senescent TIG-3 cells; 1.4 × 10 4 cells cm −2 for DXR-induced senescent RPE-1 cells. The CM to be used for sEV purification was prepared by culturing the cells in DMEM supplemented 5% EV-depleted FBS for 3 days. Replicates are biological replicates ( n =3). Error bars indicate s.d. * P

    Techniques Used: Marker, Expressing, Purification

    39) Product Images from "Dietary proanthocyanidins modulate BMAL1 acetylation, Nampt expression and NAD levels in rat liver"

    Article Title: Dietary proanthocyanidins modulate BMAL1 acetylation, Nampt expression and NAD levels in rat liver

    Journal: Scientific Reports

    doi: 10.1038/srep10954

    An oral dose of a grape seed proanthocyanidin extract (GSPE) administered at Zeitgeber Time 12 or to jet-lagged rats increases the ratio of acetylated Bmal1 in rat liver. Rats were orally gavaged with tap water (control group) or 250 mg of GSPE /kg body weight dissolved in tap water both at ( A ) ZT0 (light turned on), ( B ) ZT12 (light turned off) or ( C ) ZT6 (middle of the light period), in the case of the jet-lagged rats, which were moved to ZT12 (light turned off) after GSPE administration. Rats were sacrificed at ZT1, ZT3 or ZT6 when GSPE was administered at ZT0 and ZT13, ZT15 or ZT18 when GSPE was administered at ZT12 or to jet-lagged rats. Bmal1 RNAm levels were measured by real-time qRT-PCR and its expression was normalized to cyclophilin mRNA levels. Acetylated and total BMAL1 proteins was analyzed by Western blot and was normalized to β-actin. Acetylated and total BMAL1 protein samples were then divided to obtain the acetylated Bmal1/total Bmal1 protein ratio, shown as a percentage. Un-cropped blots/gels are presented in Supplementary Figure 1 . Each value is the mean±s.e. of the same 3 animals for all the parameters quantified. White bars, control group; colored bars, GSPE-treated groups. *Statically significant differences found by independent Student T-test (p
    Figure Legend Snippet: An oral dose of a grape seed proanthocyanidin extract (GSPE) administered at Zeitgeber Time 12 or to jet-lagged rats increases the ratio of acetylated Bmal1 in rat liver. Rats were orally gavaged with tap water (control group) or 250 mg of GSPE /kg body weight dissolved in tap water both at ( A ) ZT0 (light turned on), ( B ) ZT12 (light turned off) or ( C ) ZT6 (middle of the light period), in the case of the jet-lagged rats, which were moved to ZT12 (light turned off) after GSPE administration. Rats were sacrificed at ZT1, ZT3 or ZT6 when GSPE was administered at ZT0 and ZT13, ZT15 or ZT18 when GSPE was administered at ZT12 or to jet-lagged rats. Bmal1 RNAm levels were measured by real-time qRT-PCR and its expression was normalized to cyclophilin mRNA levels. Acetylated and total BMAL1 proteins was analyzed by Western blot and was normalized to β-actin. Acetylated and total BMAL1 protein samples were then divided to obtain the acetylated Bmal1/total Bmal1 protein ratio, shown as a percentage. Un-cropped blots/gels are presented in Supplementary Figure 1 . Each value is the mean±s.e. of the same 3 animals for all the parameters quantified. White bars, control group; colored bars, GSPE-treated groups. *Statically significant differences found by independent Student T-test (p

    Techniques Used: Quantitative RT-PCR, Expressing, Western Blot

    An oral dose of a grape seed proanthocyanidin extract (GSPE) administered at Zeitgeber Time 0 or 12 oppositely affects both Nampt expression and NAD levels in the liver. Rats were orally gavaged with tap water (control group) or 250 mg of GSPE / kg body weight dissolved in tap water both at ( A ) ZT0 (light turned on) and ( B ) ZT12 (light turned off), and rats were sacrificed at ZT1, ZT3 or ZT6 and ZT13, ZT15 or ZT18, respectively. Nampt RNAm levels were measured by real-time qRT-PCR and its expression was normalized to cyclophilin mRNA levels. Nampt protein was analyzed by Western blot and was normalized to β-actin. Un-cropped blots/gels ar e presented in Supplementary Figure 1 . NAD quantification was performed using an ELISA kit following the manufacturer’s instructions. Each value is the mean ± s.e. of the same 3 animals for all the parameters quantified. White bars, control group; colored bars, GSPE-treated groups. *Statically significant differences found by independent Student T-test (p
    Figure Legend Snippet: An oral dose of a grape seed proanthocyanidin extract (GSPE) administered at Zeitgeber Time 0 or 12 oppositely affects both Nampt expression and NAD levels in the liver. Rats were orally gavaged with tap water (control group) or 250 mg of GSPE / kg body weight dissolved in tap water both at ( A ) ZT0 (light turned on) and ( B ) ZT12 (light turned off), and rats were sacrificed at ZT1, ZT3 or ZT6 and ZT13, ZT15 or ZT18, respectively. Nampt RNAm levels were measured by real-time qRT-PCR and its expression was normalized to cyclophilin mRNA levels. Nampt protein was analyzed by Western blot and was normalized to β-actin. Un-cropped blots/gels ar e presented in Supplementary Figure 1 . NAD quantification was performed using an ELISA kit following the manufacturer’s instructions. Each value is the mean ± s.e. of the same 3 animals for all the parameters quantified. White bars, control group; colored bars, GSPE-treated groups. *Statically significant differences found by independent Student T-test (p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

    An oral dose of a grape seed proanthocyanidin extract (GSPE), administered at Zeitgeber Time 0, 12, or to jet-lagged rats, modulates HmgcoAR expression in rat liver. Rats were orally gavaged with tap water (control group) or 250 mg of GSPE / kg body weight dissolved in tap water at ( A ) ZT0 (light turned on), ( B ) ZT12 (light turned off) or ( C ) ZT6 (middle of the light period), in the case of the jet-lagged rats, which were moved to ZT12 (light turned off) after GSPE administration. Rats were sacrificed at ZT1, ZT3 or ZT6 when GSPE was administered at ZT0 and ZT13, ZT15 or ZT18 when GSPE was administered at ZT12 or to jet-lagged rats. HmgcoAR RNAm levels were measured by real-time qRT-PCR and its expression was normalized to cyclophilin mRNA levels. HmgcoAR protein was analyzed by Western blot and was normalized to β-actin. Un-cropped blots/gels are presented in Supplementary Figure 1 . White bars, control group; colored bars, GSPE-treated groups. *Statically significant differences found by independent Student T-test (p
    Figure Legend Snippet: An oral dose of a grape seed proanthocyanidin extract (GSPE), administered at Zeitgeber Time 0, 12, or to jet-lagged rats, modulates HmgcoAR expression in rat liver. Rats were orally gavaged with tap water (control group) or 250 mg of GSPE / kg body weight dissolved in tap water at ( A ) ZT0 (light turned on), ( B ) ZT12 (light turned off) or ( C ) ZT6 (middle of the light period), in the case of the jet-lagged rats, which were moved to ZT12 (light turned off) after GSPE administration. Rats were sacrificed at ZT1, ZT3 or ZT6 when GSPE was administered at ZT0 and ZT13, ZT15 or ZT18 when GSPE was administered at ZT12 or to jet-lagged rats. HmgcoAR RNAm levels were measured by real-time qRT-PCR and its expression was normalized to cyclophilin mRNA levels. HmgcoAR protein was analyzed by Western blot and was normalized to β-actin. Un-cropped blots/gels are presented in Supplementary Figure 1 . White bars, control group; colored bars, GSPE-treated groups. *Statically significant differences found by independent Student T-test (p

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    40) Product Images from "Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension"

    Article Title: Abnormal Trafficking of Endogenously Expressed BMPR2 Mutant Allelic Products in Patients with Heritable Pulmonary Arterial Hypertension

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080319

    Treatment with 4-PBA rescues signaling defects in Bmpr2 ΔEx2/+ pulmonary endothelial cells. Individual primary PECs isolates obtained from 1 wild type control and 3 Bmpr2 ΔEx2/+ mice were treated with 1mM 4-PBA for 48 hours followed by BMP2 treatment for 4 hours, as indicated. A, Western blot for phospho-Smad1/5/8 and Id1 shows 4-PBA restores phospho-Smad1/5/8 and Id1 expression in Bmpr2 ΔEx2/+ PECs to expression levels similar to BMP-stimulated WT PECs. B, Densitometry for phospho-Smad1/5/8 and C, Id1 expressed as ratio to total Smad1 and β-actin respectively. Results expressed as mean +/- SEM of three individual PEC isolates per group. One-way ANOVA with post hoc Bonferroni correction, p
    Figure Legend Snippet: Treatment with 4-PBA rescues signaling defects in Bmpr2 ΔEx2/+ pulmonary endothelial cells. Individual primary PECs isolates obtained from 1 wild type control and 3 Bmpr2 ΔEx2/+ mice were treated with 1mM 4-PBA for 48 hours followed by BMP2 treatment for 4 hours, as indicated. A, Western blot for phospho-Smad1/5/8 and Id1 shows 4-PBA restores phospho-Smad1/5/8 and Id1 expression in Bmpr2 ΔEx2/+ PECs to expression levels similar to BMP-stimulated WT PECs. B, Densitometry for phospho-Smad1/5/8 and C, Id1 expressed as ratio to total Smad1 and β-actin respectively. Results expressed as mean +/- SEM of three individual PEC isolates per group. One-way ANOVA with post hoc Bonferroni correction, p

    Techniques Used: Mouse Assay, Western Blot, Expressing

    Related Articles

    Acrylamide Gel Assay:

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    Article Snippet: .. Lysates (15 μ g) were analyzed on a 15% acrylamide gel and probed with rabbit polyclonal LC3 antibody (Cell Signaling) followed by monoclonal β -actin antibody (Sigma Aldrich, St. Louis, MO, USA) as a loading control. .. Adenovirus encoding SIRT1 (AdSIRT1) was provided by Dr. Sadoshima (Rutgers New Jersey Medical School, Newark, NJ, USA).

    Western Blot:

    Article Title: Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C *
    Article Snippet: .. Primary antibodies for immunofluorescence and Western blotting were as follows: rabbit anti-C-terminal PKCα (Epitomics); rabbit anti-β-actin (Sigma); mouse anti-Hsp70 and rat anti-Hsc70 (Enzo), and mouse anti-LC3 (Nanotools). .. Secondary antibodies for immunofluorescence and Western blotting were as follows: TRITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch), and horseradish peroxidase-conjugated goat secondary antibodies recognizing mouse, rat, or rabbit IgG (Millipore).

    Incubation:

    Article Title: FHL2 Silencing Reduces Wnt Signaling and Osteosarcoma Tumorigenesis In Vitro and In Vivo
    Article Snippet: .. Immunoblot Analysis Cell lysates were prepared and resolved on 10% SDS-PAGE as previously described were incubated with rabbit anti-FHL2 (1/1000; Abcam, Cambridge, UK), mouse anti-β-catenin (1/1000; Santa Cruz, Santa Cruz Biotechnology, CA, USA), rabbit anti-β-actin (1/2000; Sigma-Aldrich, St Quentin Fallavier, France) or mouse anti-p84 (1/1000; Abcam) antibodies. .. Membranes were then incubated with appropriate HRP-conjugated secondary antibody (1/20,000).

    SDS Page:

    Article Title: FHL2 Silencing Reduces Wnt Signaling and Osteosarcoma Tumorigenesis In Vitro and In Vivo
    Article Snippet: .. Immunoblot Analysis Cell lysates were prepared and resolved on 10% SDS-PAGE as previously described were incubated with rabbit anti-FHL2 (1/1000; Abcam, Cambridge, UK), mouse anti-β-catenin (1/1000; Santa Cruz, Santa Cruz Biotechnology, CA, USA), rabbit anti-β-actin (1/2000; Sigma-Aldrich, St Quentin Fallavier, France) or mouse anti-p84 (1/1000; Abcam) antibodies. .. Membranes were then incubated with appropriate HRP-conjugated secondary antibody (1/20,000).

    Immunofluorescence:

    Article Title: Heat Shock Proteins Regulate Activation-induced Proteasomal Degradation of the Mature Phosphorylated Form of Protein Kinase C *
    Article Snippet: .. Primary antibodies for immunofluorescence and Western blotting were as follows: rabbit anti-C-terminal PKCα (Epitomics); rabbit anti-β-actin (Sigma); mouse anti-Hsp70 and rat anti-Hsc70 (Enzo), and mouse anti-LC3 (Nanotools). .. Secondary antibodies for immunofluorescence and Western blotting were as follows: TRITC-conjugated donkey anti-rabbit (Jackson ImmunoResearch), and horseradish peroxidase-conjugated goat secondary antibodies recognizing mouse, rat, or rabbit IgG (Millipore).

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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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    99
    Millipore monoclonal anti β actin
    Zn-curc restores wild-type p53-DNA binding and transactivating activities. (A) SKBR3 and U373 cells (6x10 6 ) were plated in 150 mm dish and the day after treated with Zn-curc (100 μM) for 16 h before assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1, MDM2) or for mtp53 target promoters (MDR1, cyclin B2). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). (B) Cells (3x10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24/48 h. p53 target genes were detected by RT-PCR analysis. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to <t>β-actin</t> levels, ±SD. (C) SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc (100 μM) for 24 h, with or without p53 inhibitor pifithrin-α (PFT-α) (30 μM). p53 target genes were dtected by RT-PCR analysis. β-actin was used as control. (D) Gene expression as in (C) , was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for the indicated hours and total cell extracts were subjected to immunoblot analysis. (F) U373 cells were plated at subconfluence in 60 mm dish and the day after treated with curcumin (Curc) (50, 100 μM) for 24 h. Zn-curc (100 μM for 24 h) was used as control of p53 activation. p53 target genes were detected by RT-PCR. β-actin was used as control.
    Monoclonal Anti β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti β actin/product/Millipore
    Average 99 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti β actin - by Bioz Stars, 2020-09
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    Image Search Results


    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

    Zn-curc restores wild-type p53-DNA binding and transactivating activities. (A) SKBR3 and U373 cells (6x10 6 ) were plated in 150 mm dish and the day after treated with Zn-curc (100 μM) for 16 h before assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1, MDM2) or for mtp53 target promoters (MDR1, cyclin B2). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). (B) Cells (3x10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24/48 h. p53 target genes were detected by RT-PCR analysis. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (C) SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc (100 μM) for 24 h, with or without p53 inhibitor pifithrin-α (PFT-α) (30 μM). p53 target genes were dtected by RT-PCR analysis. β-actin was used as control. (D) Gene expression as in (C) , was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for the indicated hours and total cell extracts were subjected to immunoblot analysis. (F) U373 cells were plated at subconfluence in 60 mm dish and the day after treated with curcumin (Curc) (50, 100 μM) for 24 h. Zn-curc (100 μM for 24 h) was used as control of p53 activation. p53 target genes were detected by RT-PCR. β-actin was used as control.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells

    doi: 10.1186/1756-9966-32-72

    Figure Lengend Snippet: Zn-curc restores wild-type p53-DNA binding and transactivating activities. (A) SKBR3 and U373 cells (6x10 6 ) were plated in 150 mm dish and the day after treated with Zn-curc (100 μM) for 16 h before assayed for chromatin immunoprecipitation analysis (ChIP) with anti-p53 or anti-p73 antibodies. PCR analyses were performed on the immunoprecipitated DNA samples using primers specific for wtp53 target gene promoters (p21, Puma, p53AIP1, MDM2) or for mtp53 target promoters (MDR1, cyclin B2). A sample representing linear amplification of the total chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with non-specific immunogloblulins (No Ab). (B) Cells (3x10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24/48 h. p53 target genes were detected by RT-PCR analysis. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (C) SKBR3 and U373 cells were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc (100 μM) for 24 h, with or without p53 inhibitor pifithrin-α (PFT-α) (30 μM). p53 target genes were dtected by RT-PCR analysis. β-actin was used as control. (D) Gene expression as in (C) , was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for the indicated hours and total cell extracts were subjected to immunoblot analysis. (F) U373 cells were plated at subconfluence in 60 mm dish and the day after treated with curcumin (Curc) (50, 100 μM) for 24 h. Zn-curc (100 μM for 24 h) was used as control of p53 activation. p53 target genes were detected by RT-PCR. β-actin was used as control.

    Article Snippet: Immunoblotting was performed with the following antibodies: monoclonal anti-poly(ADP-ribose) polymerase (PARP, BD Pharmingen, CA, USA), monoclonal anti-p53 (Ab-DO1), polyclonal anti-p53 (FL393) and polyclonal anti-Bax (all from Santa Cruz Biotechnology), purified mouse anti-phospho-Histone H2AX (Ser139) (Millipore, clone JBW301; kindly provided by S. Soddu, Regina Elena National cancer Institute, Rome, Italy) and monoclonal anti-β-actin (Calbiochem).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Activation Assay

    Zn-curc impairs survival of mutant p53-carrying cells. (A) Tumor cells (4 x 10 4 ) were plated in 60 mm dish and 24 h later treated with increased amount of Zn-curc (20, 50, 100 μM). Twenty-four hours later, plates were washed with PBS and fresh medium was added. Death-resistant colonies were stained with crystal violet 14 days later. (B) Death-resistant colonies as in (A) were counted and plotted as percentage ± SD of two independent experiments performed in duplicate. (C) Cells (3 x 10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24 and 48 h. Cell viability was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. (D) Cytofluorimetric analysis of the SubG1 peak evaluated by Propidium Iodide (PI) staining (upper panel) and microscopical analysis of SKBR3 cells, mock-treated or treated with Zn-curc (100 μM) for 24 h (lower panel). Percentage of apoptotic cells is shown ± SD of two independent experiments. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-PARP (cleaved form, 87 Kd) or anti-β-actin antibodies. (F) RKO cells were treated with Zn-curc (100 μM), ZnCl 2 (100 μM) or adryamicin (ADR, 2 μg/ml) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-γH2AX (phopho-Ser139) or anti-β-actin antibodies.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells

    doi: 10.1186/1756-9966-32-72

    Figure Lengend Snippet: Zn-curc impairs survival of mutant p53-carrying cells. (A) Tumor cells (4 x 10 4 ) were plated in 60 mm dish and 24 h later treated with increased amount of Zn-curc (20, 50, 100 μM). Twenty-four hours later, plates were washed with PBS and fresh medium was added. Death-resistant colonies were stained with crystal violet 14 days later. (B) Death-resistant colonies as in (A) were counted and plotted as percentage ± SD of two independent experiments performed in duplicate. (C) Cells (3 x 10 5 ) were plated at subconfluence in 60 mm dish and the day after treated with Zn-curc for 24 and 48 h. Cell viability was measured by trypan blue exclusion assay and expressed as percentage ± SD of two independent experiments. (D) Cytofluorimetric analysis of the SubG1 peak evaluated by Propidium Iodide (PI) staining (upper panel) and microscopical analysis of SKBR3 cells, mock-treated or treated with Zn-curc (100 μM) for 24 h (lower panel). Percentage of apoptotic cells is shown ± SD of two independent experiments. (E) SKBR3 and U373 cells were treated with Zn-curc (100 μM) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-PARP (cleaved form, 87 Kd) or anti-β-actin antibodies. (F) RKO cells were treated with Zn-curc (100 μM), ZnCl 2 (100 μM) or adryamicin (ADR, 2 μg/ml) for 24 h. Equal amount of total cell extracts were subjected to immunoblot with anti-γH2AX (phopho-Ser139) or anti-β-actin antibodies.

    Article Snippet: Immunoblotting was performed with the following antibodies: monoclonal anti-poly(ADP-ribose) polymerase (PARP, BD Pharmingen, CA, USA), monoclonal anti-p53 (Ab-DO1), polyclonal anti-p53 (FL393) and polyclonal anti-Bax (all from Santa Cruz Biotechnology), purified mouse anti-phospho-Histone H2AX (Ser139) (Millipore, clone JBW301; kindly provided by S. Soddu, Regina Elena National cancer Institute, Rome, Italy) and monoclonal anti-β-actin (Calbiochem).

    Techniques: Mutagenesis, Staining, Trypan Blue Exclusion Assay

    Zn-curc reactivates mtp53 in an orthotopic U373 glioblastoma model. (A) U373MG-LUC cells (2.5x10 5 ) were injected into the brain of athymic mice and left to growth for 6 days before treating animals with Zn-curc every day for 7 days. Mock- or Zn-curc-treated U373M-derived tumors were then harvested and analysed with a fluorescent microscope that showed as diffuse fluorescence only in Zn-curc - treated tumors. (B) Quantification of tumor cell fluorescence positivity in U373-derived tumors, untreated or Zn-curc-treated, ±SD. (C) Total mRNA was extracted from harvested U373-derived tumors, untreated or Zn-curc-treated, and p53 target gene expression as well as VEGF, MDR1 and Bcl2 expression were assayed by PCR of reverse-transcribed cDNA. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD.

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: A fluorescent curcumin-based Zn(II)-complex reactivates mutant (R175H and R273H) p53 in cancer cells

    doi: 10.1186/1756-9966-32-72

    Figure Lengend Snippet: Zn-curc reactivates mtp53 in an orthotopic U373 glioblastoma model. (A) U373MG-LUC cells (2.5x10 5 ) were injected into the brain of athymic mice and left to growth for 6 days before treating animals with Zn-curc every day for 7 days. Mock- or Zn-curc-treated U373M-derived tumors were then harvested and analysed with a fluorescent microscope that showed as diffuse fluorescence only in Zn-curc - treated tumors. (B) Quantification of tumor cell fluorescence positivity in U373-derived tumors, untreated or Zn-curc-treated, ±SD. (C) Total mRNA was extracted from harvested U373-derived tumors, untreated or Zn-curc-treated, and p53 target gene expression as well as VEGF, MDR1 and Bcl2 expression were assayed by PCR of reverse-transcribed cDNA. Gene expression was measured by densitometry and plotted as fold of mRNA expression over control (Mock), normalized to β-actin levels, ±SD.

    Article Snippet: Immunoblotting was performed with the following antibodies: monoclonal anti-poly(ADP-ribose) polymerase (PARP, BD Pharmingen, CA, USA), monoclonal anti-p53 (Ab-DO1), polyclonal anti-p53 (FL393) and polyclonal anti-Bax (all from Santa Cruz Biotechnology), purified mouse anti-phospho-Histone H2AX (Ser139) (Millipore, clone JBW301; kindly provided by S. Soddu, Regina Elena National cancer Institute, Rome, Italy) and monoclonal anti-β-actin (Calbiochem).

    Techniques: Injection, Mouse Assay, Derivative Assay, Microscopy, Fluorescence, Expressing, Polymerase Chain Reaction

    ZnCl 2 induces p53 nuclear accumulation and activation in the presence of low-dose ADR. ( a ) HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), for 4-8-16-24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53 and p21. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. One representative set of blot from three independent experiments, all generating similar results, is shown here. ( b ) RKO were treated with ZnCl 2 (100 μM), for 4-8-16-24 h and the expression levels of p53 was detected by western blotting. A positive control for p53 expression in the same cells, is included (ADR 2 μg/ml for 16 h). Anti-β-actin was used as protein loading control. ( c ) RKO and HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), 8 h. Equal amounts of nuclear extracts were separated by SDS-PAGE and p53 levels detected by western blotting. Anti-Lamin A was used as protein loading control. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells

    doi: 10.1186/s13046-015-0206-x

    Figure Lengend Snippet: ZnCl 2 induces p53 nuclear accumulation and activation in the presence of low-dose ADR. ( a ) HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), for 4-8-16-24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53 and p21. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. One representative set of blot from three independent experiments, all generating similar results, is shown here. ( b ) RKO were treated with ZnCl 2 (100 μM), for 4-8-16-24 h and the expression levels of p53 was detected by western blotting. A positive control for p53 expression in the same cells, is included (ADR 2 μg/ml for 16 h). Anti-β-actin was used as protein loading control. ( c ) RKO and HCT116 were treated with low-dose ADR (0.1 μg/ml) in the presence or absence of ZnCl 2 (100 μM), 8 h. Equal amounts of nuclear extracts were separated by SDS-PAGE and p53 levels detected by western blotting. Anti-Lamin A was used as protein loading control. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here

    Article Snippet: The blotted membranes were blocked with 5 % skim milk for 1 h and then were incubated with the following primary antibodies: monoclonal anti-poly(ADP-ribose) polymerase cleaved form (PARP, BD Pharmingen, CA, USA), monoclonal anti-p53 (Ab-DO1), polyclonal antip53 (FL393), polyclonal anti-p21, monoclonal anti-MDM2 (Ab1, Santa Cruz), rabbit polyclonal anti-lamin A (all from Santa Cruz Biotechnology), rabbit polyclonal phospho-Ser46 and phospho-Ser15 (Cell Signalling and Santa Cruz), purified mouse anti-phospho-Histone H2AX (Ser139) (Millipore, clone JBW301; kindly provided by S. Soddu, Regina Elena National cancer Institute, Rome, Italy) and monoclonal anti-β-actin (Calbiochem).

    Techniques: Activation Assay, Expressing, Western Blot, Positive Control, SDS Page

    ZnCl 2 increases the low-dose ADR-induced p53 stabilization in colon cancer cells. ( a ) RKO and ( b ) HCT116, plated under the same confluence condition, were treated with increasing doses (0.1 to 2 μg/ml) of ADR in the presence or absence of ZnCl 2 (100 μM), for 24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53, γH2AX, and PARP (cleaved form). The samples derive from the same experiment and gels/blots were processed in parallel. ( c ) The phosphorylation of p53 at Ser15 and Ser46 was detected in HCT116 treated with ADR (0.1-1-2 μg/ml) in the presence or absence of ZnCl 2 (100 μM) for 24 h, by western blotting. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here. ( d ) HCT116 cells were treated with ADR (0.2 μg/ml) and ZnCl 2 (100 μM) for 24 h. After treatments, total cell extracts were immunoprecipitated with anti-p53 antibody. Western blot analysis was performed with anti-p53 and anti-MDM2 antibodies. IP: immunoprecipitation. IB: immunoblotting

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: The beneficial effect of Zinc(II) on low-dose chemotherapeutic sensitivity involves p53 activation in wild-type p53-carrying colorectal cancer cells

    doi: 10.1186/s13046-015-0206-x

    Figure Lengend Snippet: ZnCl 2 increases the low-dose ADR-induced p53 stabilization in colon cancer cells. ( a ) RKO and ( b ) HCT116, plated under the same confluence condition, were treated with increasing doses (0.1 to 2 μg/ml) of ADR in the presence or absence of ZnCl 2 (100 μM), for 24 h. Equal amounts of total cell lysates were subjected to immunoblot analysis for the detection of the expression levels of p53, γH2AX, and PARP (cleaved form). The samples derive from the same experiment and gels/blots were processed in parallel. ( c ) The phosphorylation of p53 at Ser15 and Ser46 was detected in HCT116 treated with ADR (0.1-1-2 μg/ml) in the presence or absence of ZnCl 2 (100 μM) for 24 h, by western blotting. Anti-β-actin was used as protein loading control. The samples derive from the same experiment and gels/blots were processed in parallel. The gels have been run under the same experimental conditions and one representative set of blot from three independent experiments, all generating similar results, is shown here. ( d ) HCT116 cells were treated with ADR (0.2 μg/ml) and ZnCl 2 (100 μM) for 24 h. After treatments, total cell extracts were immunoprecipitated with anti-p53 antibody. Western blot analysis was performed with anti-p53 and anti-MDM2 antibodies. IP: immunoprecipitation. IB: immunoblotting

    Article Snippet: The blotted membranes were blocked with 5 % skim milk for 1 h and then were incubated with the following primary antibodies: monoclonal anti-poly(ADP-ribose) polymerase cleaved form (PARP, BD Pharmingen, CA, USA), monoclonal anti-p53 (Ab-DO1), polyclonal antip53 (FL393), polyclonal anti-p21, monoclonal anti-MDM2 (Ab1, Santa Cruz), rabbit polyclonal anti-lamin A (all from Santa Cruz Biotechnology), rabbit polyclonal phospho-Ser46 and phospho-Ser15 (Cell Signalling and Santa Cruz), purified mouse anti-phospho-Histone H2AX (Ser139) (Millipore, clone JBW301; kindly provided by S. Soddu, Regina Elena National cancer Institute, Rome, Italy) and monoclonal anti-β-actin (Calbiochem).

    Techniques: Expressing, Western Blot, Immunoprecipitation