β actin  (Millipore)


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    Name:
    beta Actin GFP
    Description:
    iPS cell line derived with OSKM retrovirus Lines have been evaluated for exogenous gene silencing and concomitant expression of corresponding endogenous genes
    Catalog Number:
    ipsc1030
    Price:
    None
    Applications:
    The freezing media is CryoStor(R) cell cryopreservation media, Cat. No. C2874.
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    Structured Review

    Millipore β actin
    Effects of shikonin on cyclooxygenase-2 expression and on nuclear translocation of p65 and phosphorylated signal transducer and activator of transcription 3. The left panels show an example of western blot following probing with the corresponding antibody. The histograms at the right represent the data derived from the western blots following densitometry analysis. Levels were normalized against  β -actin or PARP-1 antibody. ** P
    iPS cell line derived with OSKM retrovirus Lines have been evaluated for exogenous gene silencing and concomitant expression of corresponding endogenous genes
    https://www.bioz.com/result/β actin/product/Millipore
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    Images

    1) Product Images from "Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice"

    Article Title: Beneficial Effect of Shikonin on Experimental Colitis Induced by Dextran Sulfate Sodium in Balb/C Mice

    Journal: Evidence-based Complementary and Alternative Medicine : eCAM

    doi: 10.1155/2012/271606

    Effects of shikonin on cyclooxygenase-2 expression and on nuclear translocation of p65 and phosphorylated signal transducer and activator of transcription 3. The left panels show an example of western blot following probing with the corresponding antibody. The histograms at the right represent the data derived from the western blots following densitometry analysis. Levels were normalized against  β -actin or PARP-1 antibody. ** P
    Figure Legend Snippet: Effects of shikonin on cyclooxygenase-2 expression and on nuclear translocation of p65 and phosphorylated signal transducer and activator of transcription 3. The left panels show an example of western blot following probing with the corresponding antibody. The histograms at the right represent the data derived from the western blots following densitometry analysis. Levels were normalized against β -actin or PARP-1 antibody. ** P

    Techniques Used: Expressing, Translocation Assay, Western Blot, Derivative Assay

    Expression of proinflammatory genes in peritoneal macrophages isolated from Balb/C mice. Proinflammatory gene expression levels were measured using RT-PCR, as described in  Section 2 . Representative photographs from ten independent experiments with each gene are shown.  β -Actin served as the internal control. The expression seen in the lipopolysaccharide-stimulated group (control) was standardized as 100% expression. * P
    Figure Legend Snippet: Expression of proinflammatory genes in peritoneal macrophages isolated from Balb/C mice. Proinflammatory gene expression levels were measured using RT-PCR, as described in Section 2 . Representative photographs from ten independent experiments with each gene are shown. β -Actin served as the internal control. The expression seen in the lipopolysaccharide-stimulated group (control) was standardized as 100% expression. * P

    Techniques Used: Expressing, Isolation, Mouse Assay, Reverse Transcription Polymerase Chain Reaction

    2) Product Images from "Physiological ER Stress Mediates the Differentiation of Fibroblasts"

    Article Title: Physiological ER Stress Mediates the Differentiation of Fibroblasts

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123578

    Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or anti-β-actin primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.
    Figure Legend Snippet: Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or anti-β-actin primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.

    Techniques Used: Expressing, Cell Culture, Incubation, Staining, Western Blot

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

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

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2012.00053

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

    Techniques Used: Expressing, Immunolabeling, Marker, Western Blot

    4) Product Images from "HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes"

    Article Title: HIF-1α metabolically controls: collagen synthesis and modification in chondrocytes

    Journal: Nature

    doi: 10.1038/s41586-019-0874-3

    HIF-1α silencing in PHD2-deficient chondrocytes ( a ) Expression of indicated genes in cultured chondrocytes, transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α) (n=3 biologically independent samples). ( b ) HIF-1α and Lamin A/C immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( c-f ) Oxygen consumption ( c ), glycolytic flux ( d ), energy charge ( e ) and energy status ( f ) of cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( g ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( h-i ) Proliferation ( h ) and collagen synthesis ( i ) in cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( j-k ) BiP ( j ), cleaved (c)ATF6 ( k ) and β-actin immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( l-m ) Hydroxyproline (OH-Pro) ( l ; n=6 biologically independent samples) and α-ketoglutarate (αKG) levels ( m ; n=5 biologically independent samples) in cultured chondrocytes, transduced with shScr or shHIF-1α. Data are means ± SEM. # p
    Figure Legend Snippet: HIF-1α silencing in PHD2-deficient chondrocytes ( a ) Expression of indicated genes in cultured chondrocytes, transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α) (n=3 biologically independent samples). ( b ) HIF-1α and Lamin A/C immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( c-f ) Oxygen consumption ( c ), glycolytic flux ( d ), energy charge ( e ) and energy status ( f ) of cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( g ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( h-i ) Proliferation ( h ) and collagen synthesis ( i ) in cultured chondrocytes, transduced with shScr or shHIF-1α (n=6 biologically independent samples). ( j-k ) BiP ( j ), cleaved (c)ATF6 ( k ) and β-actin immunoblot of cultured chondrocytes, transduced with shScr or shHIF-1α. Representative images of 3 independent experiments are shown. ( l-m ) Hydroxyproline (OH-Pro) ( l ; n=6 biologically independent samples) and α-ketoglutarate (αKG) levels ( m ; n=5 biologically independent samples) in cultured chondrocytes, transduced with shScr or shHIF-1α. Data are means ± SEM. # p

    Techniques Used: Expressing, Cell Culture, Transduction, shRNA

    Metabolic alterations in PHD2-deficient chondrocytes ( a ) Rhodamine labelling of mitochondria, with quantification of mitochondrial content (n= samples from 3 Phd2 chon+ and 4 Phd2 chon- mice). Yellow line denotes cell membrane. ( b-c ) Immunoblot of C-MYC ( b ), LC3-II ( c ) and β-actin levels in cultured chondrocytes. Representative images of 4 independent experiments are shown. ( d ) Oxygen consumption in cultured chondrocytes (n=9 biologically independent samples). ( e ) Pimonidazole immunostaining on neonatal (P2.5) growth plates, with a higher magnification of the boxed area and quantification of pimonidazole-positive cells within the growth plate (n=4 mice). ( f ) Glucose oxidation (GO), fatty acid oxidation (FAO) and glutamine oxidation (QO) in cultured chondrocytes (n=6 biologically independent samples). ( g ) Glucose (Glc) uptake and lactate (Lac) secretion (n=6 biologically independent samples). ( h ) Glycolytic flux (n=6 biologically independent samples). ( i ) Fractional contribution of 13 C 6 - Glc to Lac, citrate (Cit), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum) and malate (Mal) (n=6 biologically independent samples). ( j-l ) ATP content ( j ), energy charge ([ATP] + ½ [ADP] / [ATP] + [ADP] + [AMP]; k ), and energy status (ratio of ATP to AMP levels; l ) (n=6 biologically independent samples). ( m ) Apoptosis rate of cultured chondrocytes (n=4 independent experiments). ( n ) TUNEL immunostaining of neonatal growth plates (n=6 mice). ( o ) ATP production resulting from glycolysis, GO, FAO and QO in cultured chondrocytes (n=6 biologically independent samples. ( p ) Proliferation rate of cultured chondrocytes (n=4 independent experiments). ( q ) Immunoblot of Na + /K + ATPase and β-actin levels. Representative images of 3 independent experiments are shown. ( r ) Normalized Ca 2+ -rise in the cytosol of cultured chondrocytes upon stimulation with thapsigargin (TG) in the presence of EGTA (n=4 biologically independent samples). ( s ) Quantification of the Ca 2+ release from the endoplasmic reticulum (ER) upon stimulation with TG (n=4 biologically independent samples). ( t ) 45 Ca 2+ loading capacity of the ER of permeabilised chondrocytes in intracellular-like medium supplemented with 5 mM Mg/ATP and 45 Ca 2+ (n=4 biologically independent samples). ( u-v ) Total protein ( u ) and proteoglycan synthesis ( v ) (n=8 biologically independent samples). Data are means ± SEM in ( a-d, f-m, o-v ), or means ± SD in ( e, n ). *p
    Figure Legend Snippet: Metabolic alterations in PHD2-deficient chondrocytes ( a ) Rhodamine labelling of mitochondria, with quantification of mitochondrial content (n= samples from 3 Phd2 chon+ and 4 Phd2 chon- mice). Yellow line denotes cell membrane. ( b-c ) Immunoblot of C-MYC ( b ), LC3-II ( c ) and β-actin levels in cultured chondrocytes. Representative images of 4 independent experiments are shown. ( d ) Oxygen consumption in cultured chondrocytes (n=9 biologically independent samples). ( e ) Pimonidazole immunostaining on neonatal (P2.5) growth plates, with a higher magnification of the boxed area and quantification of pimonidazole-positive cells within the growth plate (n=4 mice). ( f ) Glucose oxidation (GO), fatty acid oxidation (FAO) and glutamine oxidation (QO) in cultured chondrocytes (n=6 biologically independent samples). ( g ) Glucose (Glc) uptake and lactate (Lac) secretion (n=6 biologically independent samples). ( h ) Glycolytic flux (n=6 biologically independent samples). ( i ) Fractional contribution of 13 C 6 - Glc to Lac, citrate (Cit), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum) and malate (Mal) (n=6 biologically independent samples). ( j-l ) ATP content ( j ), energy charge ([ATP] + ½ [ADP] / [ATP] + [ADP] + [AMP]; k ), and energy status (ratio of ATP to AMP levels; l ) (n=6 biologically independent samples). ( m ) Apoptosis rate of cultured chondrocytes (n=4 independent experiments). ( n ) TUNEL immunostaining of neonatal growth plates (n=6 mice). ( o ) ATP production resulting from glycolysis, GO, FAO and QO in cultured chondrocytes (n=6 biologically independent samples. ( p ) Proliferation rate of cultured chondrocytes (n=4 independent experiments). ( q ) Immunoblot of Na + /K + ATPase and β-actin levels. Representative images of 3 independent experiments are shown. ( r ) Normalized Ca 2+ -rise in the cytosol of cultured chondrocytes upon stimulation with thapsigargin (TG) in the presence of EGTA (n=4 biologically independent samples). ( s ) Quantification of the Ca 2+ release from the endoplasmic reticulum (ER) upon stimulation with TG (n=4 biologically independent samples). ( t ) 45 Ca 2+ loading capacity of the ER of permeabilised chondrocytes in intracellular-like medium supplemented with 5 mM Mg/ATP and 45 Ca 2+ (n=4 biologically independent samples). ( u-v ) Total protein ( u ) and proteoglycan synthesis ( v ) (n=8 biologically independent samples). Data are means ± SEM in ( a-d, f-m, o-v ), or means ± SD in ( e, n ). *p

    Techniques Used: Mouse Assay, Cell Culture, Immunostaining, TUNEL Assay

    Administration of α-ketoglutarate increases collagen hydroxylation and bone mass in wild-type mice ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without supplementation of dimethyl-αKG (hereafter αKG) (n=4 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with αKG (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without αKG supplementation (n=4 biologically independent samples). ( e ) Immunoblot of BiP, cleaved (c)ATF6 and β-actin in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with αKG (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with αKG, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with αKG, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with αKG, and quantification of trabecular bone volume (TBV) (n=5 mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with αKG (n=3 biologically independent samples). Data are means ± SEM in ( a-b, d-e, j ), or means ± SD in ( c, f-i, k ). # p
    Figure Legend Snippet: Administration of α-ketoglutarate increases collagen hydroxylation and bone mass in wild-type mice ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without supplementation of dimethyl-αKG (hereafter αKG) (n=4 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with αKG (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without αKG supplementation (n=4 biologically independent samples). ( e ) Immunoblot of BiP, cleaved (c)ATF6 and β-actin in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with αKG (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with αKG, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with αKG, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with αKG, and quantification of trabecular bone volume (TBV) (n=5 mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes, with or without αKG supplementation. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with αKG (n=3 biologically independent samples). Data are means ± SEM in ( a-b, d-e, j ), or means ± SD in ( c, f-i, k ). # p

    Techniques Used: Mouse Assay, Cell Culture, Staining, Immunostaining, Derivative Assay

    Phenotype of Phd2 chon- mice ( a ) Southern blot analysis showing efficient and selective recombination (black arrowhead) of the Phd2 gene in neonatal (P2.5) growth plate tissue from Phd2 chon- mice. Representative images of 3 independent experiments are shown. ( b ) Phd1 , Phd2 and Phd3 mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( c-d ) Immunoblot of PHD2 and β-actin ( c ), and of HIF-1α, HIF-2α and Lamin A/C ( d ) levels in growth plate tissue ( c ) and cultured chondrocytes ( d ). Representative images of 4 independent experiments are shown. ( e ) Quantification of tibia length (n=11 Phd2 chon+ − 8 Phd2 chon- mice), body weight (n=11 Phd2 chon+ − 8 Phd2 chon- mice), lean body mass (n=4 mice) and fat mass (n=4 mice) of adult (14-week-old) mice. ( f ) Growth-related phenotype in adult Phd2 chon- mice. ( g ) Sox9 , Col2 , Col10 , Pthrp , Ihh , Mmp9 , Mmp13 and Opn mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( h ) In situ hybridization for Col2 , Col10 , Pthrp and Ihh on neonatal growth plates (n=4 biologically independent samples; scale bar is 250 μm). ( i ) Quantification of trabecular number (Tb.N; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice) and thickness (Tb.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), cortical thickness (Ct.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), calvarial thickness (calv.Th; n=4 mice) and porosity (calv.Po; n=4 mice) in neonatal and adult mice. Nd is not determined. ( j ) Quantification of osteoblast number (N.Ob/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice), osteoblast surface (Ob.S/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice) and osteoid surface per bone surface (O.S/B.S; n=6 mice), bone formation rate (BFR; n=4 mice), mineral apposition rate (MAR; n=4 mice), osteoclast surface per bone surface (Oc.S/B.S; n=11 Phd2 chon+ − 7 Phd2 chon- mice), blood vessel number per tissue surface (N.BV/T.S; ; n=9 Phd2 chon+ − 6 Phd2 chon- mice), and serum osteocalcin (OCN; n=16 biologically independent samples), CTx-I (n=8 biologically independent samples) and CTx-II levels (n=9 biologically independent samples) in adult mice. ( k-l ) Representative images of TRAP-positive multinuclear cells formed after one week of culture ( k ) with quantification ( l ) of the number of osteoclasts formed per well (n=4 biologically independent samples; scale bar is 50 μm). Quantification was based on the number of nuclei per osteoclast. ( m ) Type I collagen (COL1) and COL2 immunostaining of the metaphysis of neonatal mice (n=8 mice). Scale bar is 100 μm. ( n ) Quantification of the cell/extracellular matrix (ECM) ratio in two zones of the growth plate (n=8 mice). Data are means ± SEM in ( c, d, l ), or means ± SD in ( b, e, g, i, j, n ). *p
    Figure Legend Snippet: Phenotype of Phd2 chon- mice ( a ) Southern blot analysis showing efficient and selective recombination (black arrowhead) of the Phd2 gene in neonatal (P2.5) growth plate tissue from Phd2 chon- mice. Representative images of 3 independent experiments are shown. ( b ) Phd1 , Phd2 and Phd3 mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( c-d ) Immunoblot of PHD2 and β-actin ( c ), and of HIF-1α, HIF-2α and Lamin A/C ( d ) levels in growth plate tissue ( c ) and cultured chondrocytes ( d ). Representative images of 4 independent experiments are shown. ( e ) Quantification of tibia length (n=11 Phd2 chon+ − 8 Phd2 chon- mice), body weight (n=11 Phd2 chon+ − 8 Phd2 chon- mice), lean body mass (n=4 mice) and fat mass (n=4 mice) of adult (14-week-old) mice. ( f ) Growth-related phenotype in adult Phd2 chon- mice. ( g ) Sox9 , Col2 , Col10 , Pthrp , Ihh , Mmp9 , Mmp13 and Opn mRNA levels in neonatal growth plates (n= independent samples from 11 Phd2 chon+ and 10 Phd2 chon- mice). ( h ) In situ hybridization for Col2 , Col10 , Pthrp and Ihh on neonatal growth plates (n=4 biologically independent samples; scale bar is 250 μm). ( i ) Quantification of trabecular number (Tb.N; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice) and thickness (Tb.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), cortical thickness (Ct.Th; for P2.5, n=10 mice, for 14 weeks, n=11 Phd2 chon+ − 8 Phd2 chon- mice), calvarial thickness (calv.Th; n=4 mice) and porosity (calv.Po; n=4 mice) in neonatal and adult mice. Nd is not determined. ( j ) Quantification of osteoblast number (N.Ob/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice), osteoblast surface (Ob.S/B.S; n=4 Phd2 chon+ − 5 Phd2 chon- mice) and osteoid surface per bone surface (O.S/B.S; n=6 mice), bone formation rate (BFR; n=4 mice), mineral apposition rate (MAR; n=4 mice), osteoclast surface per bone surface (Oc.S/B.S; n=11 Phd2 chon+ − 7 Phd2 chon- mice), blood vessel number per tissue surface (N.BV/T.S; ; n=9 Phd2 chon+ − 6 Phd2 chon- mice), and serum osteocalcin (OCN; n=16 biologically independent samples), CTx-I (n=8 biologically independent samples) and CTx-II levels (n=9 biologically independent samples) in adult mice. ( k-l ) Representative images of TRAP-positive multinuclear cells formed after one week of culture ( k ) with quantification ( l ) of the number of osteoclasts formed per well (n=4 biologically independent samples; scale bar is 50 μm). Quantification was based on the number of nuclei per osteoclast. ( m ) Type I collagen (COL1) and COL2 immunostaining of the metaphysis of neonatal mice (n=8 mice). Scale bar is 100 μm. ( n ) Quantification of the cell/extracellular matrix (ECM) ratio in two zones of the growth plate (n=8 mice). Data are means ± SEM in ( c, d, l ), or means ± SD in ( b, e, g, i, j, n ). *p

    Techniques Used: Mouse Assay, Southern Blot, Cell Culture, In Situ Hybridization, Immunostaining

    PHD2-deficient chondrocytes display enhanced glutamine metabolism ( a ) Intracellular glutamate (Glu), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum), malate (Mal) and citrate (Cit) levels in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( b ) Ratio of αKG/Suc and αKG/Fum (n=3 biologically independent samples). ( c ) GLS1 and β-actin immunoblot of cultured chondrocytes transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α). Representative images of 3 independent experiments are shown. ( d ) Immunoblot of GLS1, GLS2 and β-actin in cultured chondrocytes, compared to HeLa cells. Representative images of 3 independent experiments are shown. ( e ) Fractional contribution of 13 C 5 -glutamine (Gln) to Glu, αKG, Suc, Fum, Mal and Cit in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( f ) Citrate mass isotopomer distribution (MID) from 13 C 5 -Gln (n=3 biologically independent samples). ( g ) Relative abundance of reductive carboxylation-specific mass isotopomers of Cit, Mal and Fum (n=3 biologically independent samples). ( h ) Type II collagen (COL2) immunostaining of the tibia of neonatal (P2.5) mice treated with BPTES and/or αKG with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with BPTES, with or without αKG, and quantification of trabecular bone volume (TBV) (n=6 for Phd2 chon+ -veh, Phd2 chon+ -BPTES+αKG or Phd2 chon- -veh mice; n=5 for Phd2 chon+ -BPTES mice; and n=7 for Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with BPTES, with or without αKG (n=3 biologically independent samples). ( l ) Immunoblot of p-AMPK T172 and AMPK in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( m ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, treated with BPTES, with or without αKG (n=3 biologically independent samples). ( n ) Tibia length of mice treated with BPTES, with or without αKG (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( o ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. Data are means ± SEM in ( a-g, j, l, m, o ), or means ± SD in ( h, i, k, n ). *p
    Figure Legend Snippet: PHD2-deficient chondrocytes display enhanced glutamine metabolism ( a ) Intracellular glutamate (Glu), α-ketoglutarate (αKG), succinate (Suc), fumarate (Fum), malate (Mal) and citrate (Cit) levels in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( b ) Ratio of αKG/Suc and αKG/Fum (n=3 biologically independent samples). ( c ) GLS1 and β-actin immunoblot of cultured chondrocytes transduced with scrambled shRNA (shScr; -) or shRNA against HIF-1α (shHIF-1α). Representative images of 3 independent experiments are shown. ( d ) Immunoblot of GLS1, GLS2 and β-actin in cultured chondrocytes, compared to HeLa cells. Representative images of 3 independent experiments are shown. ( e ) Fractional contribution of 13 C 5 -glutamine (Gln) to Glu, αKG, Suc, Fum, Mal and Cit in cultured chondrocytes, with or without BPTES treatment (n=3 biologically independent samples). ( f ) Citrate mass isotopomer distribution (MID) from 13 C 5 -Gln (n=3 biologically independent samples). ( g ) Relative abundance of reductive carboxylation-specific mass isotopomers of Cit, Mal and Fum (n=3 biologically independent samples). ( h ) Type II collagen (COL2) immunostaining of the tibia of neonatal (P2.5) mice treated with BPTES and/or αKG with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with BPTES, with or without αKG, and quantification of trabecular bone volume (TBV) (n=6 for Phd2 chon+ -veh, Phd2 chon+ -BPTES+αKG or Phd2 chon- -veh mice; n=5 for Phd2 chon+ -BPTES mice; and n=7 for Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( j ) Immunoblot of HIF-1α and Lamin A/C in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( k ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with BPTES, with or without αKG (n=3 biologically independent samples). ( l ) Immunoblot of p-AMPK T172 and AMPK in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. ( m ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, treated with BPTES, with or without αKG (n=3 biologically independent samples). ( n ) Tibia length of mice treated with BPTES, with or without αKG (n=5 for Phd2 chon+ -veh or Phd2 chon- -veh mice; and n=7 for Phd2 chon+ -BPTES, Phd2 chon+ -BPTES+αKG, Phd2 chon- -BPTES or Phd2 chon- -BPTES+αKG mice). ( o ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with BPTES, with or without αKG. Representative images of 3 independent experiments are shown. Data are means ± SEM in ( a-g, j, l, m, o ), or means ± SD in ( h, i, k, n ). *p

    Techniques Used: Cell Culture, Transduction, shRNA, Immunostaining, Mouse Assay, Derivative Assay, BrdU Incorporation Assay

    Inhibition of pyruvate uptake does not affect collagen or bone properties ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without treatment with an inhibitor of monocarboxylate transporter 2 (MCT2i) (n=3 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with MCT2i (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without MCT2i treatment (n=4 biologically independent samples). ( e ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with MCT2i (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with MCT2i, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with MCT2i, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with MCT2i, and quantification of trabecular bone volume (TBV) (n=5 mice). Data are means ± SEM in ( a-b, d-e ), or means ± SD in ( c, f-i ). # p
    Figure Legend Snippet: Inhibition of pyruvate uptake does not affect collagen or bone properties ( a ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes, with or without treatment with an inhibitor of monocarboxylate transporter 2 (MCT2i) (n=3 biologically independent samples). ( b ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( c ) Tibia length of mice treated with MCT2i (n=5 mice). ( d ) Collagen synthesis in cultured chondrocytes, with or without MCT2i treatment (n=4 biologically independent samples). ( e ) BiP, cleaved (c)ATF6 and β-actin immunoblot in cultured chondrocytes treated with MCT2i. Representative images of 3 independent experiments are shown. ( f ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with MCT2i (n=5 biologically independent samples). ( g ) Safranin O staining of the tibia of mice treated with MCT2i, and quantification of the percentage Safranin O (SafO) positive matrix relative to bone volume (BV) (n=5 mice). ( h ) Type II collagen (COL2) immunostaining of the tibia of mice treated with MCT2i, with quantification of the percentage COL2-positive matrix (green) relative to bone volume (n=5 mice). GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( i ) 3D microCT models of the tibial metaphysis of mice treated with MCT2i, and quantification of trabecular bone volume (TBV) (n=5 mice). Data are means ± SEM in ( a-b, d-e ), or means ± SD in ( c, f-i ). # p

    Techniques Used: Inhibition, Cell Culture, Mouse Assay, Staining, Immunostaining

    Genetic confirmation of HIF-1α signalling and metabolic adaptations ( a-c ) Immunoblot of HIF-1α ( a ), GLS1 ( b ), PDK1 ( c ), Lamin A/C and β-actin in cultured control or PHD2-deficient (PHD2 KD ) periosteal cells, transduced with scrambled shRNA (shScr; -) or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( d ) Toluidine Blue staining of bone ossicles (n=5 biologically independent samples). Arrowheads indicate cartilage remnants (scale bar is 100 μm). ( e ) 3D CT models of bone ossicles, with quantification of the mineralized tissue volume (MV/TV) (n=5 biologically independent samples). ( f-g ) Immunoblot of GLS1 ( f ), PDK1 ( g ), and β-actin in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( h ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( i-k ) Proliferation ( i ), α-ketoglutarate (αKG) levels ( j ) and hydroxyproline (OH-Pro) content ( k ) in cultured chondrocytes, transduced with shScr or gene-specific shRNAs (n=5 biologically independent samples). Data are means ± SEM in ( a-c, f-k ), or means ± SD in ( e ). # p
    Figure Legend Snippet: Genetic confirmation of HIF-1α signalling and metabolic adaptations ( a-c ) Immunoblot of HIF-1α ( a ), GLS1 ( b ), PDK1 ( c ), Lamin A/C and β-actin in cultured control or PHD2-deficient (PHD2 KD ) periosteal cells, transduced with scrambled shRNA (shScr; -) or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( d ) Toluidine Blue staining of bone ossicles (n=5 biologically independent samples). Arrowheads indicate cartilage remnants (scale bar is 100 μm). ( e ) 3D CT models of bone ossicles, with quantification of the mineralized tissue volume (MV/TV) (n=5 biologically independent samples). ( f-g ) Immunoblot of GLS1 ( f ), PDK1 ( g ), and β-actin in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( h ) P-AMPK T172 and AMPK immunoblot with quantification of p-AMPK T172 to AMPK ratio in cultured chondrocytes, transduced with shScr or gene-specific shRNAs. Representative images of 3 independent experiments are shown. ( i-k ) Proliferation ( i ), α-ketoglutarate (αKG) levels ( j ) and hydroxyproline (OH-Pro) content ( k ) in cultured chondrocytes, transduced with shScr or gene-specific shRNAs (n=5 biologically independent samples). Data are means ± SEM in ( a-c, f-k ), or means ± SD in ( e ). # p

    Techniques Used: Cell Culture, Transduction, shRNA, Staining

    mTOR signalling and the unfolded protein response in PHD2-deficient chondrocytes ( a ) Immunoblot and quantification of phosphorylated (at Serine 2448) mTOR (p-mTOR S2448 ), mTOR, phosphorylated (at Threonine 389 and Serine 371) p70 S6 kinase (p-p70 S6K T389 and p-p70 S6K S371 ), p70 S6K, phosphorylated (at Serine 235 and 236) S6 (p-S6 S235/236 ) and S6, phosphorylated (at Threonine 37 and 46) 4E-BP1 (p-4E-BP1 T37/46 ), 4E-BP1 and β-actin in cultured chondrocytes. Representative images of 3 independent experiments are shown. ( b-c ) p-S6 S235/236 immunostaining on neonatal (P2.5) growth plates ( b ), with a higher magnification of the boxed area and quantification ( c ) of the p-S6 + area (n=6 mice). GP is growth plate, PS is primary spongiosa. ( d ) Immunoblot of p-S6 S235/236 and S6 in cultured chondrocytes. Cells were either cultured in full medium or in nutrient-deprived conditions (PBS), and then switched to full medium for indicated times. Representative images of 3 independent experiments are shown. These data show the absence of enhanced mTOR signalling. ( e ) Immunoblot and quantification of BiP, (p-)eIF2α, ATF4 and cleaved (c)ATF6 protein levels. Representative images of 3 independent experiments are shown. ( f ) Spliced Xbp-1 ( Xbp-1s ) mRNA levels in neonatal growth plates (n=8 biologically independent samples). ( g-h ) BiP and cATF6 immunostaining ( g ) of neonatal growth plates with quantification ( h ) of the percentage of positive cells (n=6 mice). Data are means ± SEM in ( a, d, e ), or means ± SD in ( c, f, h ). *p
    Figure Legend Snippet: mTOR signalling and the unfolded protein response in PHD2-deficient chondrocytes ( a ) Immunoblot and quantification of phosphorylated (at Serine 2448) mTOR (p-mTOR S2448 ), mTOR, phosphorylated (at Threonine 389 and Serine 371) p70 S6 kinase (p-p70 S6K T389 and p-p70 S6K S371 ), p70 S6K, phosphorylated (at Serine 235 and 236) S6 (p-S6 S235/236 ) and S6, phosphorylated (at Threonine 37 and 46) 4E-BP1 (p-4E-BP1 T37/46 ), 4E-BP1 and β-actin in cultured chondrocytes. Representative images of 3 independent experiments are shown. ( b-c ) p-S6 S235/236 immunostaining on neonatal (P2.5) growth plates ( b ), with a higher magnification of the boxed area and quantification ( c ) of the p-S6 + area (n=6 mice). GP is growth plate, PS is primary spongiosa. ( d ) Immunoblot of p-S6 S235/236 and S6 in cultured chondrocytes. Cells were either cultured in full medium or in nutrient-deprived conditions (PBS), and then switched to full medium for indicated times. Representative images of 3 independent experiments are shown. These data show the absence of enhanced mTOR signalling. ( e ) Immunoblot and quantification of BiP, (p-)eIF2α, ATF4 and cleaved (c)ATF6 protein levels. Representative images of 3 independent experiments are shown. ( f ) Spliced Xbp-1 ( Xbp-1s ) mRNA levels in neonatal growth plates (n=8 biologically independent samples). ( g-h ) BiP and cATF6 immunostaining ( g ) of neonatal growth plates with quantification ( h ) of the percentage of positive cells (n=6 mice). Data are means ± SEM in ( a, d, e ), or means ± SD in ( c, f, h ). *p

    Techniques Used: Cell Culture, Immunostaining, Mouse Assay

    Normalization of glucose oxidation corrects the energy deficit in PHD2-deficient chondrocytes ( a-e ) Glucose oxidation ( a ), oxygen consumption ( b ), palmitate oxidation ( c ), glutamine oxidation ( d ) and glycolytic flux ( e ) in cultured chondrocytes, with or without DCA treatment (n=6 biologically independent samples for a , n=3 biologically independent samples for b-e ). ( f ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, with or without DCA treatment (n=3 biologically independent samples). ( g ) Tibia length of mice treated with DCA (n=5 Phd2 chon+ -veh, Phd2 chon- -veh or Phd2 chon- -DCA mice - n=7 Phd2 chon+ -DCA). ( h-i ) BiP ( h ), cleaved (c)ATF6 ( i ) and β-actin immunoblot in cultured DCA-treated chondrocytes. Representative images of 3 independent experiments are shown. ( j ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with DCA, with or without BPTES (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( k ) Type II collagen (COL2) immunostaining of the tibia of mice treated with DCA, with or without BPTES with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 from Phd2 chon+ -veh mice - n=7 from Phd2 chon- -veh, Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( l ) 3D microCT models of the tibial metaphysis of mice treated with DCA, with or without BPTES, and quantification of trabecular bone volume (TBV) (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( m ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes treated with DCA, with or without BPTES (n=4 biologically independent samples). ( n ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with DCA, with or without BPTES (n=3 biologically independent samples). Data are means ± SEM in ( a-f, h-i, m ), or means ± SD in ( g, j-l, n ). # p
    Figure Legend Snippet: Normalization of glucose oxidation corrects the energy deficit in PHD2-deficient chondrocytes ( a-e ) Glucose oxidation ( a ), oxygen consumption ( b ), palmitate oxidation ( c ), glutamine oxidation ( d ) and glycolytic flux ( e ) in cultured chondrocytes, with or without DCA treatment (n=6 biologically independent samples for a , n=3 biologically independent samples for b-e ). ( f ) Proliferation, as determined by BrdU incorporation, of cultured chondrocytes, with or without DCA treatment (n=3 biologically independent samples). ( g ) Tibia length of mice treated with DCA (n=5 Phd2 chon+ -veh, Phd2 chon- -veh or Phd2 chon- -DCA mice - n=7 Phd2 chon+ -DCA). ( h-i ) BiP ( h ), cleaved (c)ATF6 ( i ) and β-actin immunoblot in cultured DCA-treated chondrocytes. Representative images of 3 independent experiments are shown. ( j ) Hydroxyproline (OH-Pro) content in neonatal growth plates of mice treated with DCA, with or without BPTES (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( k ) Type II collagen (COL2) immunostaining of the tibia of mice treated with DCA, with or without BPTES with quantification of the percentage COL2-positive matrix (green) relative to bone volume (BV) (n=5 from Phd2 chon+ -veh mice - n=7 from Phd2 chon- -veh, Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). Scale bar is 250 μm, GP is growth plate, PS is primary spongiosa, arrowheads indicate COL2 cartilage remnants. ( l ) 3D microCT models of the tibial metaphysis of mice treated with DCA, with or without BPTES, and quantification of trabecular bone volume (TBV) (n=5 from Phd2 chon+ -veh or Phd2 chon- -veh mice - n=7 from Phd2 chon+ -DCA, Phd2 chon+ -DCA+BPTES, Phd2 chon- -DCA or Phd2 chon- -DCA+BPTES mice). ( m ) Intracellular α-ketoglutarate (αKG) levels in cultured chondrocytes treated with DCA, with or without BPTES (n=4 biologically independent samples). ( n ) Relative mRNA levels of indicated genes in growth plates derived from mice treated with DCA, with or without BPTES (n=3 biologically independent samples). Data are means ± SEM in ( a-f, h-i, m ), or means ± SD in ( g, j-l, n ). # p

    Techniques Used: Cell Culture, BrdU Incorporation Assay, Mouse Assay, Immunostaining, Derivative Assay

    5) Product Images from "Superior Therapeutic Efficacy of Nanoparticle Albumin Bound Paclitaxel Over Cremophor-Bound Paclitaxel in Experimental Esophageal Adenocarcinoma"

    Article Title: Superior Therapeutic Efficacy of Nanoparticle Albumin Bound Paclitaxel Over Cremophor-Bound Paclitaxel in Experimental Esophageal Adenocarcinoma

    Journal: Translational Oncology

    doi: 10.1016/j.tranon.2018.01.022

    In-vitro comparative effects of nab-paclitaxel on the expression of apoptosis related proteins and phospho-stathmin in esophageal adenocarcinoma cells. Subconfluent monolayer of human esophageal adenocarcinoma cells OE33 (A) and OE19 (B) were treated with 5 μM of nab-paclitaxel (NPT), 5-flurouracil (5-FU), paclitaxel (PT) and carboplatin (CP) alone or in combinations for 16 hours. Equal amounts of total cell extarcts were analyzed by western blots with antibodies to cleaved caspase 3 (c-caspase 3), cleaved poly (ADP-ribose) polymerase-1 (cleaved PARP), phospho ser38 stathmin (p-Stathmin) and β-actin. Data are representative of two independent experiments with similar results.
    Figure Legend Snippet: In-vitro comparative effects of nab-paclitaxel on the expression of apoptosis related proteins and phospho-stathmin in esophageal adenocarcinoma cells. Subconfluent monolayer of human esophageal adenocarcinoma cells OE33 (A) and OE19 (B) were treated with 5 μM of nab-paclitaxel (NPT), 5-flurouracil (5-FU), paclitaxel (PT) and carboplatin (CP) alone or in combinations for 16 hours. Equal amounts of total cell extarcts were analyzed by western blots with antibodies to cleaved caspase 3 (c-caspase 3), cleaved poly (ADP-ribose) polymerase-1 (cleaved PARP), phospho ser38 stathmin (p-Stathmin) and β-actin. Data are representative of two independent experiments with similar results.

    Techniques Used: In Vitro, Expressing, Western Blot

    In-vivo comparative effects of nab-paclitaxel on the expression of apoptosis related proteins and phospho-stathmin in OE19 esophageal adenocarcinoma xenografts. Tumor lysates were prepared from OE19 xenograft tumor tissue samples obtained from tumor bearing mice after carboplatin (CP), paclitaxel (PT) or nab-paclitaxel (NPT) monotherapy or combination therapy. Tumor lysates were then analyzed by immunoblotting with antibodies to cleaved caspase 3 (c-caspase 3), cleaved poly (ADP-ribose) polymerase-1 (cleaved PARP), phospho ser38 stathmin (p-Stathmin) and β-actin. The intensity of bands was quantitated by densitometry and is represented in the bar graph after normalizing values with β-actin expression. Data are representative of pooled lysates obtained from tumors of 5 mice in each therapy group.
    Figure Legend Snippet: In-vivo comparative effects of nab-paclitaxel on the expression of apoptosis related proteins and phospho-stathmin in OE19 esophageal adenocarcinoma xenografts. Tumor lysates were prepared from OE19 xenograft tumor tissue samples obtained from tumor bearing mice after carboplatin (CP), paclitaxel (PT) or nab-paclitaxel (NPT) monotherapy or combination therapy. Tumor lysates were then analyzed by immunoblotting with antibodies to cleaved caspase 3 (c-caspase 3), cleaved poly (ADP-ribose) polymerase-1 (cleaved PARP), phospho ser38 stathmin (p-Stathmin) and β-actin. The intensity of bands was quantitated by densitometry and is represented in the bar graph after normalizing values with β-actin expression. Data are representative of pooled lysates obtained from tumors of 5 mice in each therapy group.

    Techniques Used: In Vivo, Expressing, Mouse Assay

    6) Product Images from "Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension"

    Article Title: Deletion of ADORA2B from myeloid cells dampens lung fibrosis and pulmonary hypertension

    Journal: The FASEB Journal

    doi: 10.1096/fj.14-260182

    Markers of fibrosis observed on day 33 after treatment with BLM or PBS. A ) Immunoblot for fibronectin and β -actin from whole-lung lysates from a C57BL/6 mouse treated with PBS or BLM and an Adora2B f/f -LysM Cre mouse treated with BLM. B ) Densitometry
    Figure Legend Snippet: Markers of fibrosis observed on day 33 after treatment with BLM or PBS. A ) Immunoblot for fibronectin and β -actin from whole-lung lysates from a C57BL/6 mouse treated with PBS or BLM and an Adora2B f/f -LysM Cre mouse treated with BLM. B ) Densitometry

    Techniques Used:

    Markers of alternative AAMs observed on day 33 after treatment with BLM or PBS. A ) Immunoblot for arginase-1, CD206, ADORA2B, and β -actin from whole-lung lysates from a C57BL/6 mouse treated with PBS or BLM and an Adora2B f/f -LysM Cre mouse treated
    Figure Legend Snippet: Markers of alternative AAMs observed on day 33 after treatment with BLM or PBS. A ) Immunoblot for arginase-1, CD206, ADORA2B, and β -actin from whole-lung lysates from a C57BL/6 mouse treated with PBS or BLM and an Adora2B f/f -LysM Cre mouse treated

    Techniques Used:

    7) Product Images from "An extra allele of Chk1 limits oncogene-induced replicative stress and promotes transformation"

    Article Title: An extra allele of Chk1 limits oncogene-induced replicative stress and promotes transformation

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20112147

    Alleviation of the ATR-Seckel syndrome by Chk1 Tg . (A and B) Representative images of γH2AX (A) and Ki67 (B) on the placenta and face from ATR S/S and ATR S/S / Chk1 Tg littermate embryos. Red arrows indicate cells showing a pan-nuclear γH2AX staining. Numbers indicate the mean percentage and SD of positive cells in each case ( n = 3). (C) Kaplan-Meyer curves of ATR S/S ( n = 23) and ATR S/S / Chk1 Tg ( n = 36) mice. The p-value was calculated with the Mantel-Cox log-rank test. (D) Computerized Tomography-mediated reconstruction of the heads from WT, Chk1 Tg , ATR S/S , and ATR S/S / Chk1 Tg mice. Yellow arrows indicate features that show evident rescue such as the shape of the crania (a), the micrognathia (b), and the deficient closure of the fontanelle (c). Data are representative of four independent analyses. (E) ATR, Chk1-P, and Chk1 protein levels in WT, ATR S/S , and ATR S/S / Chk1 Tg MEF treated with 0.5 mM HU for 3 h or 10 mM methyl methanesulfonate (MMS) for 4 h. β-Actin was used as a loading control. Data are representative of two independent analyses.
    Figure Legend Snippet: Alleviation of the ATR-Seckel syndrome by Chk1 Tg . (A and B) Representative images of γH2AX (A) and Ki67 (B) on the placenta and face from ATR S/S and ATR S/S / Chk1 Tg littermate embryos. Red arrows indicate cells showing a pan-nuclear γH2AX staining. Numbers indicate the mean percentage and SD of positive cells in each case ( n = 3). (C) Kaplan-Meyer curves of ATR S/S ( n = 23) and ATR S/S / Chk1 Tg ( n = 36) mice. The p-value was calculated with the Mantel-Cox log-rank test. (D) Computerized Tomography-mediated reconstruction of the heads from WT, Chk1 Tg , ATR S/S , and ATR S/S / Chk1 Tg mice. Yellow arrows indicate features that show evident rescue such as the shape of the crania (a), the micrognathia (b), and the deficient closure of the fontanelle (c). Data are representative of four independent analyses. (E) ATR, Chk1-P, and Chk1 protein levels in WT, ATR S/S , and ATR S/S / Chk1 Tg MEF treated with 0.5 mM HU for 3 h or 10 mM methyl methanesulfonate (MMS) for 4 h. β-Actin was used as a loading control. Data are representative of two independent analyses.

    Techniques Used: Staining, Mouse Assay

    Generation of a Chk1 Tg strain. (A) Region including Chk1 that was subcloned from the mouse genome. (B) Southern blot with an internal Chk1 probe, illustrating the presence of an integration site (8 kb) on the Chk1 Tg strain. The 5-kb band corresponds to the endogenous Chk1 . (C) Representative picture of 4-mo-old WT and Chk1 Tg littermates. (D) Weight distribution of 1-mo-old WT and Chk1 Tg mice. (E) Chk1 Western blot in testis, spleen, and purified B cells after a 2-d stimulation with lipopolysaccharide from WT and Chk1 Tg mice. Data are representative of four independent analyses. (F) ATR, Chk1-P, Chk1, RPA-P, and Rad17-P Western blot in WT and Chk1 Tg littermate MEF, either untreated (C) or upon treatment with 2 mM HU for 3 h or 10 Gy IR for 1 h. Data are representative of two independent analyses. (G) Chk1-P, RPA-P, and Rad17-P Western blot in WT and Chk1 Tg littermate MEF upon treatment with 2 mM HU for 3 h in the presence or absence of 1 µM of ATR inhibitor (ATRi; ETP-46464). Data are representative of two independent analyses. β-Actin was used as a loading control in all Western blots. In D, center lines indicate mean values.
    Figure Legend Snippet: Generation of a Chk1 Tg strain. (A) Region including Chk1 that was subcloned from the mouse genome. (B) Southern blot with an internal Chk1 probe, illustrating the presence of an integration site (8 kb) on the Chk1 Tg strain. The 5-kb band corresponds to the endogenous Chk1 . (C) Representative picture of 4-mo-old WT and Chk1 Tg littermates. (D) Weight distribution of 1-mo-old WT and Chk1 Tg mice. (E) Chk1 Western blot in testis, spleen, and purified B cells after a 2-d stimulation with lipopolysaccharide from WT and Chk1 Tg mice. Data are representative of four independent analyses. (F) ATR, Chk1-P, Chk1, RPA-P, and Rad17-P Western blot in WT and Chk1 Tg littermate MEF, either untreated (C) or upon treatment with 2 mM HU for 3 h or 10 Gy IR for 1 h. Data are representative of two independent analyses. (G) Chk1-P, RPA-P, and Rad17-P Western blot in WT and Chk1 Tg littermate MEF upon treatment with 2 mM HU for 3 h in the presence or absence of 1 µM of ATR inhibitor (ATRi; ETP-46464). Data are representative of two independent analyses. β-Actin was used as a loading control in all Western blots. In D, center lines indicate mean values.

    Techniques Used: Southern Blot, Mouse Assay, Western Blot, Purification, Recombinase Polymerase Amplification

    8) Product Images from "Epigenetic Changes in Mitochondrial Superoxide Dismutase in the Retina and the Development of Diabetic Retinopathy"

    Article Title: Epigenetic Changes in Mitochondrial Superoxide Dismutase in the Retina and the Development of Diabetic Retinopathy

    Journal: Diabetes

    doi: 10.2337/db10-0133

    H4K20me3 and acetyl H3K9 at sod2 , and evaluation of ChIP controls in retinal endothelial cells. A and B : H4K20me3 and acetyl H3K9 at the sod2 promoter and enhancer were measured by ChIP assay with SYBR green-based real-time q-PCR. Rabbit IgG served as a negative antibody control (indicated as ^). C : ChIP controls were verified by PCR. Crosslinked cells were immunoprecipitated with acetyl H3K9 or TFIIB antibody or normal rabbit IgG. The sod2 promoter occupied by acetyl H3K9 and the β-actin promoter occupied by TFIIB were amplified in purified ChIP-DNA. For the ChIP assay, the positive and negative controls were β-actin promoter occupied by TFIIB and the sod2 promoter occupied by IgG, respectively. The internal control included the input. 5 and 20, cells incubated in 5 mmol/L glucose or 20 mmol/L glucose; +Mn, cells transfected with sod2 plasmid, followed incubation in 20 mmol/L glucose for 4 days; and Mann, cells incubated in 20 mmol/L mannitol instead of 20 mmol/L glucose. * P
    Figure Legend Snippet: H4K20me3 and acetyl H3K9 at sod2 , and evaluation of ChIP controls in retinal endothelial cells. A and B : H4K20me3 and acetyl H3K9 at the sod2 promoter and enhancer were measured by ChIP assay with SYBR green-based real-time q-PCR. Rabbit IgG served as a negative antibody control (indicated as ^). C : ChIP controls were verified by PCR. Crosslinked cells were immunoprecipitated with acetyl H3K9 or TFIIB antibody or normal rabbit IgG. The sod2 promoter occupied by acetyl H3K9 and the β-actin promoter occupied by TFIIB were amplified in purified ChIP-DNA. For the ChIP assay, the positive and negative controls were β-actin promoter occupied by TFIIB and the sod2 promoter occupied by IgG, respectively. The internal control included the input. 5 and 20, cells incubated in 5 mmol/L glucose or 20 mmol/L glucose; +Mn, cells transfected with sod2 plasmid, followed incubation in 20 mmol/L glucose for 4 days; and Mann, cells incubated in 20 mmol/L mannitol instead of 20 mmol/L glucose. * P

    Techniques Used: Chromatin Immunoprecipitation, SYBR Green Assay, Polymerase Chain Reaction, Immunoprecipitation, Amplification, Purification, Incubation, Transfection, Plasmid Preparation

    Retinal H4K20me3 and acetyl H3K9 at sod2 , methylation of H4K20, and the expression of SUV420h2: the levels of acetyl H3K9 ( A ) and H4K20me3 ( B ) at the sod2 promoter and enhancer were determined using the ChIP technique. Retinal chromatins were immunoprecipitated with H4K20me3 or acetyl H3K9 antibody, and the sod2 promoter and enhancer region were amplified. The q-PCR value in each immunoprecipitate was normalized to the Ct value from the input sample using the ΔΔCt method. C : The protein expression of methylated H4K20 was detected by Western blot technique using antibodies specific for mono-, di-, and trimethyl H4K20 using histone H4 as the loading control. D : Gene expression of SUV420h2 was detected by q-PCR and analyzed by the ΔΔCt method. The housekeeping gene was β-actin. Fold-changes were calculated by setting the mean fraction of normal rats as 1. E : Protein expression of SUV420h2 was quantified by Western blot, and β-actin was used as a loading protein. Values are represented as mean ± SD of five to six rats in each group. * P
    Figure Legend Snippet: Retinal H4K20me3 and acetyl H3K9 at sod2 , methylation of H4K20, and the expression of SUV420h2: the levels of acetyl H3K9 ( A ) and H4K20me3 ( B ) at the sod2 promoter and enhancer were determined using the ChIP technique. Retinal chromatins were immunoprecipitated with H4K20me3 or acetyl H3K9 antibody, and the sod2 promoter and enhancer region were amplified. The q-PCR value in each immunoprecipitate was normalized to the Ct value from the input sample using the ΔΔCt method. C : The protein expression of methylated H4K20 was detected by Western blot technique using antibodies specific for mono-, di-, and trimethyl H4K20 using histone H4 as the loading control. D : Gene expression of SUV420h2 was detected by q-PCR and analyzed by the ΔΔCt method. The housekeeping gene was β-actin. Fold-changes were calculated by setting the mean fraction of normal rats as 1. E : Protein expression of SUV420h2 was quantified by Western blot, and β-actin was used as a loading protein. Values are represented as mean ± SD of five to six rats in each group. * P

    Techniques Used: Methylation, Expressing, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Polymerase Chain Reaction, Western Blot

    Gene expression of sod2 in rat retina. RNA was isolated from the retina using TRIzol reagent, and 1 μg RNA was converted to single-stranded cDNA and quantified spectrophotometerically. Gene expression was measured in a 90-ng cDNA template using the ABI-7500 sequence detection system. Each sample was analyzed in triplicate, and the data were normalized to β-actin expression in each sample. Fold-change relative to age-matched normal rats was calculated using the ΔΔCt method. Results are from five or more rats in each group and are represented as mean ± SD. * P
    Figure Legend Snippet: Gene expression of sod2 in rat retina. RNA was isolated from the retina using TRIzol reagent, and 1 μg RNA was converted to single-stranded cDNA and quantified spectrophotometerically. Gene expression was measured in a 90-ng cDNA template using the ABI-7500 sequence detection system. Each sample was analyzed in triplicate, and the data were normalized to β-actin expression in each sample. Fold-change relative to age-matched normal rats was calculated using the ΔΔCt method. Results are from five or more rats in each group and are represented as mean ± SD. * P

    Techniques Used: Expressing, Isolation, Sequencing

    9) Product Images from "Differential Inhibition of Protein Translation Machinery by Curcumin in Normal, Immortalized and Malignant Oral Epithelial Cells"

    Article Title: Differential Inhibition of Protein Translation Machinery by Curcumin in Normal, Immortalized and Malignant Oral Epithelial Cells

    Journal: Cancer prevention research (Philadelphia, Pa.)

    doi: 10.1158/1940-6207.CAPR-09-0076

    Effect of curcumin on cap-dependent translation. (a) Schematic diagram of Ren-HCV-FF mRNA; Graphical representation of the effects of various doses of curcumin on cap-dependent translation (Renilla luciferase) and HCV IRES-mediated translation (Firefly luciferase) in intact (b) NOM9-CT, and (c) UMSCC22B cells. Luciferase activity in treated cells is expressed relative to cells treated with vehicle (DMSO) alone. The results are the average of three experiments; error bars, s.e.m., significantly different (NOM9-CT, p=0.0005 and UMSCC22B, p=0.0004) compared with DMSO-treated control by one-way ANOVA; (d) NOM9-CT and (e) UMSCC22B cells were treated for 8 hrs with DMSO or various doses of curcumin. The whole cell lysates were resolved on 10% PAGE in the presence of SDS and subjected to Western blot analysis using antibodies against cyclin-D1, c-Myc, ODC, Yes and β-actin.
    Figure Legend Snippet: Effect of curcumin on cap-dependent translation. (a) Schematic diagram of Ren-HCV-FF mRNA; Graphical representation of the effects of various doses of curcumin on cap-dependent translation (Renilla luciferase) and HCV IRES-mediated translation (Firefly luciferase) in intact (b) NOM9-CT, and (c) UMSCC22B cells. Luciferase activity in treated cells is expressed relative to cells treated with vehicle (DMSO) alone. The results are the average of three experiments; error bars, s.e.m., significantly different (NOM9-CT, p=0.0005 and UMSCC22B, p=0.0004) compared with DMSO-treated control by one-way ANOVA; (d) NOM9-CT and (e) UMSCC22B cells were treated for 8 hrs with DMSO or various doses of curcumin. The whole cell lysates were resolved on 10% PAGE in the presence of SDS and subjected to Western blot analysis using antibodies against cyclin-D1, c-Myc, ODC, Yes and β-actin.

    Techniques Used: Luciferase, Activity Assay, Polyacrylamide Gel Electrophoresis, Western Blot

    10) Product Images from "Myeloid Heme Oxygenase-1 Regulates the Acute Inflammatory Response to Zymosan in the Mouse Air Pouch"

    Article Title: Myeloid Heme Oxygenase-1 Regulates the Acute Inflammatory Response to Zymosan in the Mouse Air Pouch

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2018/5053091

    Cell migration into the MAP. (a) Cell numbers in the exudate, mean ± SD ( n  = 6). (b) Immunoblot for HO-1 expression in cells of the exudate. Representative images and rate of HO-1/ β -actin;  ++ P
    Figure Legend Snippet: Cell migration into the MAP. (a) Cell numbers in the exudate, mean ± SD ( n = 6). (b) Immunoblot for HO-1 expression in cells of the exudate. Representative images and rate of HO-1/ β -actin; ++ P

    Techniques Used: Migration, Expressing

    11) Product Images from "GSK3? Is Involved in JNK2-Mediated ?-Catenin Inhibition"

    Article Title: GSK3? Is Involved in JNK2-Mediated ?-Catenin Inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006640

    JNK2 deficiency caused upregulation of β-catenin and its downstream target CDK4, as well as upregulation of GSK3β phosphorylation in JNK2-/- mouse intestinal epithelial cells, compared to those in JNK2+/+ mice. Each lane represents one mouse. β-actin served as loading control.
    Figure Legend Snippet: JNK2 deficiency caused upregulation of β-catenin and its downstream target CDK4, as well as upregulation of GSK3β phosphorylation in JNK2-/- mouse intestinal epithelial cells, compared to those in JNK2+/+ mice. Each lane represents one mouse. β-actin served as loading control.

    Techniques Used: Mouse Assay

    Active JNK2 downregulated β-catenin expression, inhibited its transcriptional activity and reduced GSK3β phosphorylation. (A) Active JNK2 suppressed β-catenin expression and GSK3β phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-β-catenin together with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK, p-c-Jun, phospho-Ser 9 GSK3β, and GSK3β. β-actin served as loading control. (B) Active JNK2 reduced GSK3β phosphorylation and downregulated β-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of β-catenin, p-JNK, and phospho-Ser 9 GSK3β. β-actin served as loading control. (C) Active JNK inhibited β-catenin-mediated transcriptional activity of TCF. HEK293T cells were co-transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, pcDNA3-HA-β-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.
    Figure Legend Snippet: Active JNK2 downregulated β-catenin expression, inhibited its transcriptional activity and reduced GSK3β phosphorylation. (A) Active JNK2 suppressed β-catenin expression and GSK3β phosphorylation in HEK293T cells. HEK293T cells were transfected with pcDNA3-HA-β-catenin together with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin, p-JNK, p-c-Jun, phospho-Ser 9 GSK3β, and GSK3β. β-actin served as loading control. (B) Active JNK2 reduced GSK3β phosphorylation and downregulated β-catenin expression in human lung cancer cell line A549. A549 cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of β-catenin, p-JNK, and phospho-Ser 9 GSK3β. β-actin served as loading control. (C) Active JNK inhibited β-catenin-mediated transcriptional activity of TCF. HEK293T cells were co-transfected with pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, pcDNA3-HA-β-catenin, TOPFLASH (TOP) or FOPFLASH (FOP), and Renilla. 48 h after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.

    Techniques Used: Expressing, Activity Assay, Transfection, Luciferase, Standard Deviation

    Active JNK2 downregulated β-catenin expression and inhibited its transcriptional activity in a dose-dependent manner. (A) Activated JNK2 reduced β-catenin protein level in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-β-catenin along with different amounts of pcDNA3-Flag-MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin and p-JNK. β-actin served as loading control. (B) Activated JNK2 inhibited β-catenin-mediated transcriptional activity of TCF in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-β-catenin, TOPFLASH, Renilla, along with different amounts of pcDNA3-Flag-MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.
    Figure Legend Snippet: Active JNK2 downregulated β-catenin expression and inhibited its transcriptional activity in a dose-dependent manner. (A) Activated JNK2 reduced β-catenin protein level in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-β-catenin along with different amounts of pcDNA3-Flag-MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for immunoblotting analysis to detect the alterations of HA-β-catenin and p-JNK. β-actin served as loading control. (B) Activated JNK2 inhibited β-catenin-mediated transcriptional activity of TCF in a dose-dependent manner. HEK293T cells were co-transfected with pcDNA3-HA-β-catenin, TOPFLASH, Renilla, along with different amounts of pcDNA3-Flag-MKK7-JNK2, as indicated. Forty-eight hours after transfection, cells were harvested for luciferase activity assay. Each bar represents the mean ± standard deviation (SD) for triplicated samples.

    Techniques Used: Expressing, Activity Assay, Transfection, Luciferase, Standard Deviation

    Active JNK2-mediated β-catenin degradation occurred through the proteasome system and GSK3β. (A) HEK293T cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Forty-four hours after transfection, 25 µM MG132 was added to the indicated samples (lane 2 and 4). Four hours later cells were harvested for immunoblotting analysis to detect the expression of HA-β-catenin and p-JNK. (B) Blocking GSK3β activity by LiCl reduced β-catenin expression inhibition by activated JNK2. pcDNA3-HA-β-catenin was transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Thirty-six hours after transfection, half of the cultures were treated overnight with 30 mM LiCl (lane 2 and 4) and then harvested for immunoblotting analysis to detect the expression of HA-β-catenin, phospho-Ser-9 GSK3β, and p-JNK. (C) Mutant β-catenin was resistant to activated JNK2 induced degradation. Wild-type β-catenin (HA- β-catenin) (lanes 1 and 2) or various β-catenin mutants (HA-S33F β-catenin, lanes 3 and 4; HA-S33Y β-catenin, lanes 5 and 6; HA-S37A β-catenin, lanes 7 and 8) were transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 2,4,6,8) or empty vector (lanes 1,3,5,7). 48 hours after transfection, cells were harvested for immunoblotting analysis to determine the protein levels of HA-β-catenin. β-actin served as loading control.
    Figure Legend Snippet: Active JNK2-mediated β-catenin degradation occurred through the proteasome system and GSK3β. (A) HEK293T cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Forty-four hours after transfection, 25 µM MG132 was added to the indicated samples (lane 2 and 4). Four hours later cells were harvested for immunoblotting analysis to detect the expression of HA-β-catenin and p-JNK. (B) Blocking GSK3β activity by LiCl reduced β-catenin expression inhibition by activated JNK2. pcDNA3-HA-β-catenin was transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 3 and 4) or empty vector (lane 1 and 2). Thirty-six hours after transfection, half of the cultures were treated overnight with 30 mM LiCl (lane 2 and 4) and then harvested for immunoblotting analysis to detect the expression of HA-β-catenin, phospho-Ser-9 GSK3β, and p-JNK. (C) Mutant β-catenin was resistant to activated JNK2 induced degradation. Wild-type β-catenin (HA- β-catenin) (lanes 1 and 2) or various β-catenin mutants (HA-S33F β-catenin, lanes 3 and 4; HA-S33Y β-catenin, lanes 5 and 6; HA-S37A β-catenin, lanes 7 and 8) were transfected into HEK293T cells along with pcDNA3-Flag-MKK7-JNK2 (lane 2,4,6,8) or empty vector (lanes 1,3,5,7). 48 hours after transfection, cells were harvested for immunoblotting analysis to determine the protein levels of HA-β-catenin. β-actin served as loading control.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Blocking Assay, Activity Assay, Inhibition, Mutagenesis

    12) Product Images from "Digibind attenuates cytokine TNF?-induced endothelial inflammatory response: potential benefit role of Digibind in preeclampsia"

    Article Title: Digibind attenuates cytokine TNF?-induced endothelial inflammatory response: potential benefit role of Digibind in preeclampsia

    Journal: Journal of perinatology : official journal of the California Perinatal Association

    doi: 10.1038/jp.2008.222

    Effects of tumor necrosis factor-α (TNFα) on extracellular signal-regulated kinase (ERK), pERK and Na + /K + -ATPase β1 expressions in ECs. β-Actin expression was used as control. TNFα dose-dependently induced pERK upregulation and Na + /K + -ATPase β1 downregulation in ECs. The blots are representative from three independent experiments.
    Figure Legend Snippet: Effects of tumor necrosis factor-α (TNFα) on extracellular signal-regulated kinase (ERK), pERK and Na + /K + -ATPase β1 expressions in ECs. β-Actin expression was used as control. TNFα dose-dependently induced pERK upregulation and Na + /K + -ATPase β1 downregulation in ECs. The blots are representative from three independent experiments.

    Techniques Used: Expressing

    13) Product Images from "Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis"

    Article Title: Switching of Pyruvate Kinase Isoform L to M2 Promotes Metabolic Reprogramming in Hepatocarcinogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115036

    PKM2 promoted HCC growth in vitro through regulating aerobic glycolysis and ROS levels. (A) Two stable PKM2 knockdown clones were generated in MHCC-97L and SMMC-7721 cells. Expression of PKM2, PKM1, and β actin were evaluated by Western Blots. (B) Knockdown of PKM2 by two independent sequences consistently reduced HCC cell proliferation rate by cell counting. (C) Knockdown of PKM2 reduced lactate accumulation in multiple HCC cell lines. (D) Colorimetric assay showed that knockdown of PKM2 reduced the glucose consumption rate of multiple HCC cell lines. (E) Glucose uptake in HCC cells was confirmed with 2-NBDG staining. (F) Knockdown of PKM2 increased ROS accumulation in multiple HCC cells. (G) Knockdown of PKM2 decreased NADPH level in SMMC-7721 cells. Values were normalized to NTC of the according cell lines. * P
    Figure Legend Snippet: PKM2 promoted HCC growth in vitro through regulating aerobic glycolysis and ROS levels. (A) Two stable PKM2 knockdown clones were generated in MHCC-97L and SMMC-7721 cells. Expression of PKM2, PKM1, and β actin were evaluated by Western Blots. (B) Knockdown of PKM2 by two independent sequences consistently reduced HCC cell proliferation rate by cell counting. (C) Knockdown of PKM2 reduced lactate accumulation in multiple HCC cell lines. (D) Colorimetric assay showed that knockdown of PKM2 reduced the glucose consumption rate of multiple HCC cell lines. (E) Glucose uptake in HCC cells was confirmed with 2-NBDG staining. (F) Knockdown of PKM2 increased ROS accumulation in multiple HCC cells. (G) Knockdown of PKM2 decreased NADPH level in SMMC-7721 cells. Values were normalized to NTC of the according cell lines. * P

    Techniques Used: In Vitro, Clone Assay, Generated, Expressing, Western Blot, Cell Counting, Colorimetric Assay, Staining

    14) Product Images from "Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt"

    Article Title: Activation of volume-sensitive outwardly rectifying chloride channel by ROS contributes to ER stress and cardiac contractile dysfunction: involvement of CHOP through Wnt

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2014.479

    VSOR Cl − channel blockers rescue tunicamycin-induced ER stress in cardiomyocytes. Cardiomyocytes were treated with Tm (100 ng/ml) in the presence or absence of DIDS or DCPIB for 24 h. ( a ) Representative images of immunostaining for GRP78 (Green). Nuclei were labeled with DAPI; n =5 for each group. ( b ) qRT-PCR assay for XBP1S expression. β -Actin served as a loading control. * P
    Figure Legend Snippet: VSOR Cl − channel blockers rescue tunicamycin-induced ER stress in cardiomyocytes. Cardiomyocytes were treated with Tm (100 ng/ml) in the presence or absence of DIDS or DCPIB for 24 h. ( a ) Representative images of immunostaining for GRP78 (Green). Nuclei were labeled with DAPI; n =5 for each group. ( b ) qRT-PCR assay for XBP1S expression. β -Actin served as a loading control. * P

    Techniques Used: Immunostaining, Labeling, Quantitative RT-PCR, Expressing

    VSOR Cl − channel blockers rescue tunicamycin-induced ER stress in vivo . VSOR Cl − channel was blocked with DIDS and DCPIB for 24 h before assessment of ER stress. ( a ) qRT-PCR assay for XBP1S expression. β -actin served as a loading control. * P
    Figure Legend Snippet: VSOR Cl − channel blockers rescue tunicamycin-induced ER stress in vivo . VSOR Cl − channel was blocked with DIDS and DCPIB for 24 h before assessment of ER stress. ( a ) qRT-PCR assay for XBP1S expression. β -actin served as a loading control. * P

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

    15) Product Images from "Chlorpromazine protects against acetaminophen-induced liver injury in mice by modulating autophagy and c-Jun N-terminal kinase activation ☆"

    Article Title: Chlorpromazine protects against acetaminophen-induced liver injury in mice by modulating autophagy and c-Jun N-terminal kinase activation ☆

    Journal: Liver research

    doi: 10.1016/j.livres.2019.01.004

    CQ co-treatment partially ameliorates the protective effects of CPZ against APAP-induced liver injury. (A) Mice were treated i.p . with APAP (500 mg/kg) and CQ (60 mg/kg) and 2 h later these mice were further treated with CPZ (6 mg/kg, i.p .) for another 4 h. (B) Serum ALT levels were analyzed. (C) Representative immunoblot of LC3. β-Actin was used as loading control. (D) Densitometry of (C) ( N = 3). Results are presented as the mean ± SEM. Student’s t -test, * P
    Figure Legend Snippet: CQ co-treatment partially ameliorates the protective effects of CPZ against APAP-induced liver injury. (A) Mice were treated i.p . with APAP (500 mg/kg) and CQ (60 mg/kg) and 2 h later these mice were further treated with CPZ (6 mg/kg, i.p .) for another 4 h. (B) Serum ALT levels were analyzed. (C) Representative immunoblot of LC3. β-Actin was used as loading control. (D) Densitometry of (C) ( N = 3). Results are presented as the mean ± SEM. Student’s t -test, * P

    Techniques Used: Mouse Assay

    CPZ does not affect APAP metabolism. Mice were treated i.p . with APAP (500 mg/kg) and CPZ (6mg/kg) simultaneously, and livers were collected at 0.5, 2, 6 and 24h respectively. (A) Representative immunoblot of CYP2E1 and APAP-ADs in liver collected at 6 h after treatment. β-actin was used as loading control. (B) Densitometry of (A). (C) Total GSH and GSSG were measured, and GSSG/GSH ratios were calculated ( N = 3—4). Data are presented as the mean ± SEM. Student’s t -test, * P
    Figure Legend Snippet: CPZ does not affect APAP metabolism. Mice were treated i.p . with APAP (500 mg/kg) and CPZ (6mg/kg) simultaneously, and livers were collected at 0.5, 2, 6 and 24h respectively. (A) Representative immunoblot of CYP2E1 and APAP-ADs in liver collected at 6 h after treatment. β-actin was used as loading control. (B) Densitometry of (A). (C) Total GSH and GSSG were measured, and GSSG/GSH ratios were calculated ( N = 3—4). Data are presented as the mean ± SEM. Student’s t -test, * P

    Techniques Used: Mouse Assay

    CPZ co-treatment decreases hepatic levels of p62 and LC3-II. Mice were treated i.p. with APAP (500 mg/kg) and CPZ (6 mg/kg) simultaneously, and livers were collected at 6 h. (A) Representative immunoblot of liver p62, LC3, p-4EBP1 and total 4EBP1. β-actin was used as loading control. (B) Densitometry of (A) ( N = 3—4). Abbreviations: CPZ, chlorpromazine; APAP, acetaminophen; i.p ., intraperitoneally; LC3, microtubule-associated protein 1 light chain 3; 4EBP1, eukaryotic initiation factor 4E-binding protein 1.
    Figure Legend Snippet: CPZ co-treatment decreases hepatic levels of p62 and LC3-II. Mice were treated i.p. with APAP (500 mg/kg) and CPZ (6 mg/kg) simultaneously, and livers were collected at 6 h. (A) Representative immunoblot of liver p62, LC3, p-4EBP1 and total 4EBP1. β-actin was used as loading control. (B) Densitometry of (A) ( N = 3—4). Abbreviations: CPZ, chlorpromazine; APAP, acetaminophen; i.p ., intraperitoneally; LC3, microtubule-associated protein 1 light chain 3; 4EBP1, eukaryotic initiation factor 4E-binding protein 1.

    Techniques Used: Mouse Assay, Binding Assay

    16) Product Images from "ARYL HYDROCARBON RECEPTOR DEFICIENCY PROTECTS MICE FROM DIET-INDUCED ADIPOSITY AND METABOLIC DISORDERS THROUGH INCREASED ENERGY EXPENDITURE"

    Article Title: ARYL HYDROCARBON RECEPTOR DEFICIENCY PROTECTS MICE FROM DIET-INDUCED ADIPOSITY AND METABOLIC DISORDERS THROUGH INCREASED ENERGY EXPENDITURE

    Journal: International journal of obesity (2005)

    doi: 10.1038/ijo.2015.63

    AhR deficiency improves HFD-induced insulin resistance, hyperglycemia and hyperinsulinemia ( A and B ) Glucose tolerance test (GTT) after 10 weeks of NCD or HFD feeding ( A ) and insulin tolerance test (ITT) after 12 weeks of NCD or HFD feeding ( B ) in WT, AhR −/− and AhR +/− mice. ( C ) Fasted and fed blood glucose were measured from the same mice in ( A and B ). (NCD, n=7-14; HFD, WT, n=17, AhR −/− , n=13, AhR +/− , n=13 for A, B, and C). ( D ) After 14 weeks of HFD feeding, some mice received an injection of insulin (0.75U/kg) through inferior vena cava, tissues were harvested at pre- injection and post-injection (3 min for liver, 5 min for gastrocnemius muscle, 7 min for eWAT). Western- blot analysis was performed to assess insulin-stimulated phospho-Akt (Ser308 and Ser473), total Akt and β-actin were used to normalize. n=3-5 for each group, Representative blots were shown for each group. *p
    Figure Legend Snippet: AhR deficiency improves HFD-induced insulin resistance, hyperglycemia and hyperinsulinemia ( A and B ) Glucose tolerance test (GTT) after 10 weeks of NCD or HFD feeding ( A ) and insulin tolerance test (ITT) after 12 weeks of NCD or HFD feeding ( B ) in WT, AhR −/− and AhR +/− mice. ( C ) Fasted and fed blood glucose were measured from the same mice in ( A and B ). (NCD, n=7-14; HFD, WT, n=17, AhR −/− , n=13, AhR +/− , n=13 for A, B, and C). ( D ) After 14 weeks of HFD feeding, some mice received an injection of insulin (0.75U/kg) through inferior vena cava, tissues were harvested at pre- injection and post-injection (3 min for liver, 5 min for gastrocnemius muscle, 7 min for eWAT). Western- blot analysis was performed to assess insulin-stimulated phospho-Akt (Ser308 and Ser473), total Akt and β-actin were used to normalize. n=3-5 for each group, Representative blots were shown for each group. *p

    Techniques Used: Mouse Assay, Injection, Western Blot

    AhR deficiency alters chronic inflammation and adipokine levels Mice were sacrificed after 14 weeks of NCD or HFD feeding. eWAT and liver were harvested and total RNA and protein were extracted. Real-time PCR analysis for the macrophage marker genes CD68 and F4/80; cytokines TNFα, IL-1β, IL-6, and IL10 in eWAT ( A ) and liver ( B ). Levels of mRNA in ( A ) and ( B ) are expressed as a ratio of HFD to NCD within the same genotype to accentuate changes associated with HFD feeding. Western blot analysis was performed for p-JNK and IκBα expression in HFD-fed mice in eWAT ( C ) and liver ( D ). Transcript levels in eWAT were determined for the adipokines, leptin ( E ) and adiponectin ( F ). PCR data in ( A ), ( B ), ( E ) and ( F ) were normalized against the amount of B2M; Western blot data in ( C ) and ( D ) were normalized to β-Actin. n=8-12 for each group, * p
    Figure Legend Snippet: AhR deficiency alters chronic inflammation and adipokine levels Mice were sacrificed after 14 weeks of NCD or HFD feeding. eWAT and liver were harvested and total RNA and protein were extracted. Real-time PCR analysis for the macrophage marker genes CD68 and F4/80; cytokines TNFα, IL-1β, IL-6, and IL10 in eWAT ( A ) and liver ( B ). Levels of mRNA in ( A ) and ( B ) are expressed as a ratio of HFD to NCD within the same genotype to accentuate changes associated with HFD feeding. Western blot analysis was performed for p-JNK and IκBα expression in HFD-fed mice in eWAT ( C ) and liver ( D ). Transcript levels in eWAT were determined for the adipokines, leptin ( E ) and adiponectin ( F ). PCR data in ( A ), ( B ), ( E ) and ( F ) were normalized against the amount of B2M; Western blot data in ( C ) and ( D ) were normalized to β-Actin. n=8-12 for each group, * p

    Techniques Used: Mouse Assay, Real-time Polymerase Chain Reaction, Marker, Western Blot, Expressing, Polymerase Chain Reaction

    17) Product Images from "The Iron Chelator, Dp44mT, Effectively Inhibits Human Oral Squamous Cell Carcinoma Cell Growth in Vitro and in Vivo"

    Article Title: The Iron Chelator, Dp44mT, Effectively Inhibits Human Oral Squamous Cell Carcinoma Cell Growth in Vitro and in Vivo

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17091435

    Anti-proliferative effects of Dp44mT and the effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in OECM-1 cells. ( A ) OECM-1 cells were treated with various concentrations of Dp44mt as indicated for 24 h and growth inhibitory effect was determined by the CyQUANT cell proliferation assay. The data shown in each bar chart represent the mean percentage ± SE of cells in each dose of the iron chelators treatment and are compared with the control solvent-treated group ( n = 8). The OECM-1 cells were treated with various concentrations of Dp44mt ( B ), DFO ( C ), and deferasirox ( D ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group, + p
    Figure Legend Snippet: Anti-proliferative effects of Dp44mT and the effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in OECM-1 cells. ( A ) OECM-1 cells were treated with various concentrations of Dp44mt as indicated for 24 h and growth inhibitory effect was determined by the CyQUANT cell proliferation assay. The data shown in each bar chart represent the mean percentage ± SE of cells in each dose of the iron chelators treatment and are compared with the control solvent-treated group ( n = 8). The OECM-1 cells were treated with various concentrations of Dp44mt ( B ), DFO ( C ), and deferasirox ( D ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group, + p

    Techniques Used: CyQUANT Assay, Proliferation Assay, Western Blot, Produced

    Effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in SAS cells. The SAS cells were treated with various concentrations of Dp44mt ( A ), DFO ( B ), and deferasirox ( C ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group ( D – F ), + p
    Figure Legend Snippet: Effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in SAS cells. The SAS cells were treated with various concentrations of Dp44mt ( A ), DFO ( B ), and deferasirox ( C ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group ( D – F ), + p

    Techniques Used: Western Blot, Produced

    18) Product Images from "Overexpression of Inhibitor of Growth 4 Enhances Radiosensitivity in Non-Small Cell Lung Cancer Cell Line SPC-A1"

    Article Title: Overexpression of Inhibitor of Growth 4 Enhances Radiosensitivity in Non-Small Cell Lung Cancer Cell Line SPC-A1

    Journal: Technology in Cancer Research & Treatment

    doi: 10.1177/1533034616656315

    Expression analysis of Bcl-2, Bax, activated caspase-3 and PCNA after pcDNA3.1-ING4 transfection. A. Western blotting analysis for determining expression of Bcl-2, Bax, activated casepase-3 in SPC-A1 cells after pcDNA3.1-ING4 transfection in vitro . β-actin protein was used as an internal control. B. Immunostaining was used to detect PCNA, Bcl-2 and Bax-2 in the xenografts of nude mice in vivo .
    Figure Legend Snippet: Expression analysis of Bcl-2, Bax, activated caspase-3 and PCNA after pcDNA3.1-ING4 transfection. A. Western blotting analysis for determining expression of Bcl-2, Bax, activated casepase-3 in SPC-A1 cells after pcDNA3.1-ING4 transfection in vitro . β-actin protein was used as an internal control. B. Immunostaining was used to detect PCNA, Bcl-2 and Bax-2 in the xenografts of nude mice in vivo .

    Techniques Used: Expressing, Transfection, Western Blot, In Vitro, Immunostaining, Mouse Assay, In Vivo

    Expression of ING4 in human NSCLC cancers. A. Relative expression levels of ING4 protein were detected in 28 cases of NSCLC tissue samples and corresponding non-tumor lung tissues by immunoblotting. β-actin protein was used as an internal control. N, non-tumor tissues; T, NSCLC tissues. The data is representive of 3 independent experiments. B. Immunohistochemistry analysis of the expression of the ING4 protein in NSCLC tissues. Positive expression of ING4 was determined as brown staining of the nucleus and cytoplasm. Immunostaining of ING4 was strongly positive in corresponding non-tumor lung tissues (a, b), and was positive in the cytoplasm but not the nucleus in lung adenocarcinomas (c, d) or almost negative in squamous cell carcinomas (e, f). Original magnification, ×100.
    Figure Legend Snippet: Expression of ING4 in human NSCLC cancers. A. Relative expression levels of ING4 protein were detected in 28 cases of NSCLC tissue samples and corresponding non-tumor lung tissues by immunoblotting. β-actin protein was used as an internal control. N, non-tumor tissues; T, NSCLC tissues. The data is representive of 3 independent experiments. B. Immunohistochemistry analysis of the expression of the ING4 protein in NSCLC tissues. Positive expression of ING4 was determined as brown staining of the nucleus and cytoplasm. Immunostaining of ING4 was strongly positive in corresponding non-tumor lung tissues (a, b), and was positive in the cytoplasm but not the nucleus in lung adenocarcinomas (c, d) or almost negative in squamous cell carcinomas (e, f). Original magnification, ×100.

    Techniques Used: Expressing, Immunohistochemistry, Staining, Immunostaining

    RT-PCR and western blot analysis of ING4 expression in SPC-A1/pcDNA3.1-ING4 compared with SPC-A1/pcDNA3.1. A. The epression of ING4 mRNA was upregulated in SPC-A1/pcDNA3.1-ING4 by RT-PCR and β-actin was used as an internal control. B. ING4 protein expression was upregulated in SPCA1/pcDNA3.1-ING4 by Western blotting and β-actin was used as an internal control.
    Figure Legend Snippet: RT-PCR and western blot analysis of ING4 expression in SPC-A1/pcDNA3.1-ING4 compared with SPC-A1/pcDNA3.1. A. The epression of ING4 mRNA was upregulated in SPC-A1/pcDNA3.1-ING4 by RT-PCR and β-actin was used as an internal control. B. ING4 protein expression was upregulated in SPCA1/pcDNA3.1-ING4 by Western blotting and β-actin was used as an internal control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

    19) Product Images from "Overexpression of Protein Kinase Mζ in the Hippocampus Enhances Long-Term Potentiation and Long-Term Contextual But Not Cued Fear Memory in Rats"

    Article Title: Overexpression of Protein Kinase Mζ in the Hippocampus Enhances Long-Term Potentiation and Long-Term Contextual But Not Cued Fear Memory in Rats

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3600-15.2016

    Quantitative analysis of protein expression in hippocampal tissue 5 weeks after AAV-mediated PKMζ-WT and PKM KD overexpression in rats. A , Representative Western blot image. PKMζ is blotted at 51 kDa and β-actin at 37 kDa was loaded as a normalizing protein. Hippocampal PKMζ-WT and PKMζ-KD are 1.5-fold increased compared with PBS-injected animals. B , Plot of six to seven hippocampi per group, normalized to endogenous PKMζ level in PBS-treated rats. * ⧸ ** p
    Figure Legend Snippet: Quantitative analysis of protein expression in hippocampal tissue 5 weeks after AAV-mediated PKMζ-WT and PKM KD overexpression in rats. A , Representative Western blot image. PKMζ is blotted at 51 kDa and β-actin at 37 kDa was loaded as a normalizing protein. Hippocampal PKMζ-WT and PKMζ-KD are 1.5-fold increased compared with PBS-injected animals. B , Plot of six to seven hippocampi per group, normalized to endogenous PKMζ level in PBS-treated rats. * ⧸ ** p

    Techniques Used: Expressing, Over Expression, Western Blot, Injection

    20) Product Images from "Virus-induced unfolded protein response attenuates anti-viral defenses via phosphorylation-dependent degradation of the Type I interferon receptor"

    Article Title: Virus-induced unfolded protein response attenuates anti-viral defenses via phosphorylation-dependent degradation of the Type I interferon receptor

    Journal: Cell host & microbe

    doi: 10.1016/j.chom.2008.11.008

    ER stress promotes IFNAR1 ubiquitination and degradation in a ligand/Jak-independent manner A. Levels of endogenous IFNAR1 in 293T cells pre-treated or not with methylamine HCl (MA, 20mM) for 1 h and then treated with TG (1μM) for indicated time were analyzed by IP-IB. Levels of β-actin in whole cell lysates are also shown. B. Cells harboring the WT Tyk2 (WT-5) or the kinase dead Tyk2 (KR-2) were treated with TG as indicated and ubiquitination and levels of endogenous IFNAR1 were analyzed by IP-IB. Aliquots of whole cell lysates were also analyzed by IB using anti-β-actin antibody (lower panel). C. 293T cells were pre-treated or not with MA for 1 h and then treated with cycloheximide (Chx, 50μg/ml) alone or together with TG (1μM) for indicated times. Levels of endogenous IFNAR1 were analyzed by IP-IB. Levels of c-Jun and β-actin in whole cell lysates were also determined by IB using indicated antibodies. D. Ubiquitination of Flag-tagged IFNAR1 co-expressed with the indicated shRNA constructs in 293T cells was analyzed by IP using anti-Flag antibody followed by IB using anti-ubiquitin and anti-Flag antibodies as indicated. Aliquots of whole cell lysates were also analyzed by IB using anti-β-actin antibody (lower panel). E. PERK fl/fl MEFs that either underwent acute deletion of PERK (Cre) or not (Mock) were treated with 1μM of TG (together with Chx, 10μg/ml) for 45 min as indicated. Endogenous mouse IFNAR1 was analyzed by IP-IB using the indicated antibodies. Ig: heavy chain immunoglobulins. Whole cell lysates were also subjected to IB analysis to determine levels of phosphorylated β-catenin and eIF2α as well as total levels of PERK and eIF2α using respective antibodies. NS: non-specific band. F. Mouse Flag-IFNAR1 expressed in PERK fl/fl MEFs that either underwent acute deletion of PERK (Cre) or not (Mock) were analyzed by IB using anti-Flag antibody. Levels of PERK are shown in lower panel. NS: non-specific band that serves as a loading control.
    Figure Legend Snippet: ER stress promotes IFNAR1 ubiquitination and degradation in a ligand/Jak-independent manner A. Levels of endogenous IFNAR1 in 293T cells pre-treated or not with methylamine HCl (MA, 20mM) for 1 h and then treated with TG (1μM) for indicated time were analyzed by IP-IB. Levels of β-actin in whole cell lysates are also shown. B. Cells harboring the WT Tyk2 (WT-5) or the kinase dead Tyk2 (KR-2) were treated with TG as indicated and ubiquitination and levels of endogenous IFNAR1 were analyzed by IP-IB. Aliquots of whole cell lysates were also analyzed by IB using anti-β-actin antibody (lower panel). C. 293T cells were pre-treated or not with MA for 1 h and then treated with cycloheximide (Chx, 50μg/ml) alone or together with TG (1μM) for indicated times. Levels of endogenous IFNAR1 were analyzed by IP-IB. Levels of c-Jun and β-actin in whole cell lysates were also determined by IB using indicated antibodies. D. Ubiquitination of Flag-tagged IFNAR1 co-expressed with the indicated shRNA constructs in 293T cells was analyzed by IP using anti-Flag antibody followed by IB using anti-ubiquitin and anti-Flag antibodies as indicated. Aliquots of whole cell lysates were also analyzed by IB using anti-β-actin antibody (lower panel). E. PERK fl/fl MEFs that either underwent acute deletion of PERK (Cre) or not (Mock) were treated with 1μM of TG (together with Chx, 10μg/ml) for 45 min as indicated. Endogenous mouse IFNAR1 was analyzed by IP-IB using the indicated antibodies. Ig: heavy chain immunoglobulins. Whole cell lysates were also subjected to IB analysis to determine levels of phosphorylated β-catenin and eIF2α as well as total levels of PERK and eIF2α using respective antibodies. NS: non-specific band. F. Mouse Flag-IFNAR1 expressed in PERK fl/fl MEFs that either underwent acute deletion of PERK (Cre) or not (Mock) were analyzed by IB using anti-Flag antibody. Levels of PERK are shown in lower panel. NS: non-specific band that serves as a loading control.

    Techniques Used: shRNA, Construct

    Viral infection promotes phosphorylation-dependent ubiquitination and downregulation of IFNAR1 in a Tyk2-independent and S535/526-dependent manner. A. Ubiquitination, phosphorylation and total levels of endogenous IFNAR1 from KR-2 cells infected with VSV (for 16, 18 and 20 h) were analyzed by IP using anti-IFNAR1 antibody followed by IB using the indicated antibodies. Viral protein accumulation is shown by the levels of VSV-M. B. Levels of endogenous IFNAR1 in the lysates from Huh7 cells (parental or harboring either a full length or subgenomic HCV) were analyzed by IP-IB. Levels of β-actin in the lysates aliquots are also shown. C. Endogenous IFNAR1 proteins were immunopurified from the indicated cells (as in panel C) and loaded onto the gel to yield comparable levels of total IFNAR1 (lower panel). Phosphorylation of IFNAR1 was then analyzed by IB using indicated antibody (upper panel). D. MEFs from IFNAR1-/- mice were stably reconstituted with murine Flag-IFNAR1 (either wild type or S526A mutant) and then infected with VSV (for 16-18 h.) Levels of IFNAR1, VSV-M and β-actin were analyzed by IB. E. EB-derived cells of WT (WT/WT) or mutant (S526A/WT) genotype were infected (or not) with VSV for 12 h and lysed. Under these conditions, levels of VSV-M become saturated at 10hr post-infection (data not shown). Levels of endogenous mouse IFNAR1 were determined by IP-IB. Levels of β-actin and VSV-M in the lysates were also determined.
    Figure Legend Snippet: Viral infection promotes phosphorylation-dependent ubiquitination and downregulation of IFNAR1 in a Tyk2-independent and S535/526-dependent manner. A. Ubiquitination, phosphorylation and total levels of endogenous IFNAR1 from KR-2 cells infected with VSV (for 16, 18 and 20 h) were analyzed by IP using anti-IFNAR1 antibody followed by IB using the indicated antibodies. Viral protein accumulation is shown by the levels of VSV-M. B. Levels of endogenous IFNAR1 in the lysates from Huh7 cells (parental or harboring either a full length or subgenomic HCV) were analyzed by IP-IB. Levels of β-actin in the lysates aliquots are also shown. C. Endogenous IFNAR1 proteins were immunopurified from the indicated cells (as in panel C) and loaded onto the gel to yield comparable levels of total IFNAR1 (lower panel). Phosphorylation of IFNAR1 was then analyzed by IB using indicated antibody (upper panel). D. MEFs from IFNAR1-/- mice were stably reconstituted with murine Flag-IFNAR1 (either wild type or S526A mutant) and then infected with VSV (for 16-18 h.) Levels of IFNAR1, VSV-M and β-actin were analyzed by IB. E. EB-derived cells of WT (WT/WT) or mutant (S526A/WT) genotype were infected (or not) with VSV for 12 h and lysed. Under these conditions, levels of VSV-M become saturated at 10hr post-infection (data not shown). Levels of endogenous mouse IFNAR1 were determined by IP-IB. Levels of β-actin and VSV-M in the lysates were also determined.

    Techniques Used: Infection, Mouse Assay, Stable Transfection, Mutagenesis, Derivative Assay

    21) Product Images from "Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin Bound-Paclitaxel in Pancreatic Ductal Adenocarcinoma"

    Article Title: Bisphosphonates Inhibit Stellate Cell Activity and Enhance Antitumor Effects of Nanoparticle Albumin Bound-Paclitaxel in Pancreatic Ductal Adenocarcinoma

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-14-0028

    PSCs are inactivated by Pam and ZA. Immunoblotting of α-SMA (a marker of PSC activation) and unprenylated Rap1A from PSCs treated with Pam (A) or ZA (B). MCP-1 levels are reduced by Pam (C) and ZA(D) in PSCs. Type I collagen expression levels are reduced in PSCs treated with Pam (E) or ZA (F). Immunoblots were scanned using an Alpha Imager densitometer for quantification. Data was normalized to β-actin and vinculin. Error bars represent SD of the mean of triplicates. *, P
    Figure Legend Snippet: PSCs are inactivated by Pam and ZA. Immunoblotting of α-SMA (a marker of PSC activation) and unprenylated Rap1A from PSCs treated with Pam (A) or ZA (B). MCP-1 levels are reduced by Pam (C) and ZA(D) in PSCs. Type I collagen expression levels are reduced in PSCs treated with Pam (E) or ZA (F). Immunoblots were scanned using an Alpha Imager densitometer for quantification. Data was normalized to β-actin and vinculin. Error bars represent SD of the mean of triplicates. *, P

    Techniques Used: Marker, Activation Assay, Expressing, Western Blot

    22) Product Images from "The contribution of N2O3 to the cytotoxicity of the nitric oxide donor DETA/NO: an emerging role for S-nitrosylation"

    Article Title: The contribution of N2O3 to the cytotoxicity of the nitric oxide donor DETA/NO: an emerging role for S-nitrosylation

    Journal: Bioscience Reports

    doi: 10.1042/BSR20120120

    Biotin switch assay to determine the extent of protein S-nitrosylation following exposure to NO • and ONOO − RSNO profile in MDA-MB-231 cells following the treatment with ONOO − (1 mM), AS (1 mM), or LD 50 concentrations of DETA/NO under normoxia or 0.1% hypoxia ( * ), (2) or MDA-MB-231 cells treated with DETA/NO±sodium azide. (3) The S-nitrosylated total protein profile in nuclear extracts following the treatment with LD 50 concentrations of DETA/NO. Whole cell or nuclear lysates were subjected to the biotin switch method, biotin labelled nitrosoproteins were separated on SDS/PAGE (10% gel) and detected by Western blot analysis with an anti-biotin antibody. In parallel, β-actin or H2B protein levels served as a loading control. Images shown are the representative blots of three independent experiments. Arrows on the left point to visible bands representing the molecular mass of S-nitrosylated proteins.
    Figure Legend Snippet: Biotin switch assay to determine the extent of protein S-nitrosylation following exposure to NO • and ONOO − RSNO profile in MDA-MB-231 cells following the treatment with ONOO − (1 mM), AS (1 mM), or LD 50 concentrations of DETA/NO under normoxia or 0.1% hypoxia ( * ), (2) or MDA-MB-231 cells treated with DETA/NO±sodium azide. (3) The S-nitrosylated total protein profile in nuclear extracts following the treatment with LD 50 concentrations of DETA/NO. Whole cell or nuclear lysates were subjected to the biotin switch method, biotin labelled nitrosoproteins were separated on SDS/PAGE (10% gel) and detected by Western blot analysis with an anti-biotin antibody. In parallel, β-actin or H2B protein levels served as a loading control. Images shown are the representative blots of three independent experiments. Arrows on the left point to visible bands representing the molecular mass of S-nitrosylated proteins.

    Techniques Used: Biotin Switch Assay, Multiple Displacement Amplification, SDS Page, Western Blot

    23) Product Images from "Transforming Growth Factor-? Requires Its Target Plasminogen Activator Inhibitor-1 for Cytostatic Activity *Transforming Growth Factor-? Requires Its Target Plasminogen Activator Inhibitor-1 for Cytostatic Activity * S⃞"

    Article Title: Transforming Growth Factor-? Requires Its Target Plasminogen Activator Inhibitor-1 for Cytostatic Activity *Transforming Growth Factor-? Requires Its Target Plasminogen Activator Inhibitor-1 for Cytostatic Activity * S⃞

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M803341200

    SMAD signaling and target activation in TGFβ-resistant PAI-1 kd HaCaT cells and MEFs. a , analysis of SMAD pathway activation in HaCaT cells. Western blot of activated phospho-SMAD2 versus normal SMAD2 in untreated and TGFβ-bypassing cells expressing depicted constructs or a nonfunctional shRNA control. β-Actin is a loading control. b , analysis of TGFβ-targets PAI-1, p21 CIP1 , and p15 INK4b expression by QRT-PCR in TGFβ-bypassing HaCaT cells expressing depicted constructs. c , analysis of SMAD-pathway activation in MEFs. Western blot of activated phospho ( p )-SMAD2 versus normal SMAD2 in untreated and TGFβ-bypassing cells expressing depicted constructs and nonfunctional shRNA expressing control cells. β-Actin is a loading control. d , mRNA analysis of TGFβ-target PAI-1 in TGFβ-bypassing MEFs expressing depicted constructs versus a nonfunctional shRNA expressing and non-treated control.
    Figure Legend Snippet: SMAD signaling and target activation in TGFβ-resistant PAI-1 kd HaCaT cells and MEFs. a , analysis of SMAD pathway activation in HaCaT cells. Western blot of activated phospho-SMAD2 versus normal SMAD2 in untreated and TGFβ-bypassing cells expressing depicted constructs or a nonfunctional shRNA control. β-Actin is a loading control. b , analysis of TGFβ-targets PAI-1, p21 CIP1 , and p15 INK4b expression by QRT-PCR in TGFβ-bypassing HaCaT cells expressing depicted constructs. c , analysis of SMAD-pathway activation in MEFs. Western blot of activated phospho ( p )-SMAD2 versus normal SMAD2 in untreated and TGFβ-bypassing cells expressing depicted constructs and nonfunctional shRNA expressing control cells. β-Actin is a loading control. d , mRNA analysis of TGFβ-target PAI-1 in TGFβ-bypassing MEFs expressing depicted constructs versus a nonfunctional shRNA expressing and non-treated control.

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

    Constitutive PI3K-PKB signaling induces bypass of the cytostatic response to TGFβ in human keratinocytes. a , loss of PAI-1 retains PI3K-PKB signaling in HaCaT cells in the presence of TGFβ. Western blot analysis of activated phospho ( p )-PKB versus normal PKB in TGFβ-bypassing cells expressing a nonfunctional shRNA control or PAI-1 kd or uPA overexpressing constructs, respectively. β-Actin is a loading control. b , colony formation assay in HaCaT cells overexpressing the indicated constructs treated with TGFβ for 7 days. A nonfunctional shRNA is used as a negative control. Also shown are phase contrast images of the cells after 7 days in culture in the presence of TGFβ. ca , constitutively. c , Western blot analysis of activated phospho-PKB versus normal PKB and PTEN in TGFβ-bypassing cells expressing a nonfunctional shRNA control, PTEN kd , or constitutively active PI3K or PKB overexpressing construct. The asterisk indicates an aspecific background band. β-Actin is a loading control.
    Figure Legend Snippet: Constitutive PI3K-PKB signaling induces bypass of the cytostatic response to TGFβ in human keratinocytes. a , loss of PAI-1 retains PI3K-PKB signaling in HaCaT cells in the presence of TGFβ. Western blot analysis of activated phospho ( p )-PKB versus normal PKB in TGFβ-bypassing cells expressing a nonfunctional shRNA control or PAI-1 kd or uPA overexpressing constructs, respectively. β-Actin is a loading control. b , colony formation assay in HaCaT cells overexpressing the indicated constructs treated with TGFβ for 7 days. A nonfunctional shRNA is used as a negative control. Also shown are phase contrast images of the cells after 7 days in culture in the presence of TGFβ. ca , constitutively. c , Western blot analysis of activated phospho-PKB versus normal PKB and PTEN in TGFβ-bypassing cells expressing a nonfunctional shRNA control, PTEN kd , or constitutively active PI3K or PKB overexpressing construct. The asterisk indicates an aspecific background band. β-Actin is a loading control.

    Techniques Used: Western Blot, Expressing, shRNA, Construct, Colony Assay, Negative Control

    24) Product Images from "Oxidized Low-Density Lipoprotein Induces Apoptosis in Endothelial Progenitor Cells by Inactivating the Phosphoinositide 3-Kinase/Akt Pathway"

    Article Title: Oxidized Low-Density Lipoprotein Induces Apoptosis in Endothelial Progenitor Cells by Inactivating the Phosphoinositide 3-Kinase/Akt Pathway

    Journal: Journal of Vascular Research

    doi: 10.1159/000313879

    Effects of myrAkt on nLDL- and oxLDL-induced apoptosis in wild type EPC. a Effects of Ad-DLE1 (control transduction) and Admiralty (constitutively active Akt transduction) on p-Akt, total Akt, p-GSK3α, total GSK3α and β-actin expression in wild-type EPC. b EPC cultures, transducer with either Ad-DLE1 (control; empty vector) or Ad-myrAkt (constitutively active Akt) were exposed to nLDL (10 μg/ml) or oxLDL (10 μg/ml), or oxLDL (10 μg/ml) in serum-free medium. Apoptosis was determined by TUNEL staining. Data are expressed as the percentage of TUNEL positive cells with respect to the total number of cells (DAPI staining) per high power field. Data are presented as mean ± SD, n = 3; + p
    Figure Legend Snippet: Effects of myrAkt on nLDL- and oxLDL-induced apoptosis in wild type EPC. a Effects of Ad-DLE1 (control transduction) and Admiralty (constitutively active Akt transduction) on p-Akt, total Akt, p-GSK3α, total GSK3α and β-actin expression in wild-type EPC. b EPC cultures, transducer with either Ad-DLE1 (control; empty vector) or Ad-myrAkt (constitutively active Akt) were exposed to nLDL (10 μg/ml) or oxLDL (10 μg/ml), or oxLDL (10 μg/ml) in serum-free medium. Apoptosis was determined by TUNEL staining. Data are expressed as the percentage of TUNEL positive cells with respect to the total number of cells (DAPI staining) per high power field. Data are presented as mean ± SD, n = 3; + p

    Techniques Used: Transduction, Expressing, Plasmid Preparation, TUNEL Assay, Staining

    Effects of SOD, L -NAME, epicatechin and FeTTPs on the ratio of p-Akt/total Akt in wild-type EPC. EPC cultures were exposed to nLDL (10 μg/ml) or oxLDL (10 μg/ml), or oxLDL (10 μg/ml) in the presence of SOD (100 U/ml), L -NAME (0.5 m M ), epicatechin (100 μ M ) or FeTPPs (2.5 μ M ) in serum-free medium. A control well was treated with serum-free medium alone. Density of the Akt and p-Akt bands was first corrected with respect to β-actin (not shown) and then the ratio of p-Akt/total Akt expression calculated. The ratio in the control group was assigned a value of 1.0. Data are presented as mean ± SD, n = 3; + p
    Figure Legend Snippet: Effects of SOD, L -NAME, epicatechin and FeTTPs on the ratio of p-Akt/total Akt in wild-type EPC. EPC cultures were exposed to nLDL (10 μg/ml) or oxLDL (10 μg/ml), or oxLDL (10 μg/ml) in the presence of SOD (100 U/ml), L -NAME (0.5 m M ), epicatechin (100 μ M ) or FeTPPs (2.5 μ M ) in serum-free medium. A control well was treated with serum-free medium alone. Density of the Akt and p-Akt bands was first corrected with respect to β-actin (not shown) and then the ratio of p-Akt/total Akt expression calculated. The ratio in the control group was assigned a value of 1.0. Data are presented as mean ± SD, n = 3; + p

    Techniques Used: Expressing

    Effects of SOD, L -NAME, epicatechin and FeTPPs on nitrotyrosine expression and the ratio of the PI3K p85 and p110 subunits in wild-type EPC. EPC cultures were exposed to nLDL (10 μg/ml) or oxLDL (10 μg/ml), or oxLDL (10 μg/ml) in the presence of SOD (100 U/ml), L -NAME (0.5 m M ), epicatechin (100 μ M ) or FeTPPs (2.5 μ M ) in serum-free medium. A control well was treated with serum-free medium alone. a The p110 Akt subunit was isolated by immunoprecipitation; the expression of p110 and nitrotyrosine was determined. Note the essential absence of nitrotyrosine. This set of Western blots is representative of 3 separate experiments. b The p85 Akt subunit was isolated by immunoprecipitation; the expression of p110 and nitrotyrosine were determined. Density of the nitrotyrosine and PI3K p85 subunit bands were first corrected with respect to β-actin and then the ratio of nitrotyrosine/p85 expression calculated. The ratio of the control group was assigned a value of 1.0. Data are presented as mean ± SD, n = 3; + p
    Figure Legend Snippet: Effects of SOD, L -NAME, epicatechin and FeTPPs on nitrotyrosine expression and the ratio of the PI3K p85 and p110 subunits in wild-type EPC. EPC cultures were exposed to nLDL (10 μg/ml) or oxLDL (10 μg/ml), or oxLDL (10 μg/ml) in the presence of SOD (100 U/ml), L -NAME (0.5 m M ), epicatechin (100 μ M ) or FeTPPs (2.5 μ M ) in serum-free medium. A control well was treated with serum-free medium alone. a The p110 Akt subunit was isolated by immunoprecipitation; the expression of p110 and nitrotyrosine was determined. Note the essential absence of nitrotyrosine. This set of Western blots is representative of 3 separate experiments. b The p85 Akt subunit was isolated by immunoprecipitation; the expression of p110 and nitrotyrosine were determined. Density of the nitrotyrosine and PI3K p85 subunit bands were first corrected with respect to β-actin and then the ratio of nitrotyrosine/p85 expression calculated. The ratio of the control group was assigned a value of 1.0. Data are presented as mean ± SD, n = 3; + p

    Techniques Used: Expressing, Isolation, Immunoprecipitation, Western Blot

    Time- and dose-response relationships between oxLDL and the expression of Akt and p-Akt in wild-type EPC. a EPC cultures were exposed to nLDL (10 μg/ml) or oxLDL (1–50 μg/ml) in serum-free medium; the control well was treated with serum-free medium alone. Density of the Akt and p-Akt bands were first corrected with respect to β-actin (not shown) and then the ratio of p-Akt/total Akt expression calculated. The ratio of the wild-type group was assigned a value of 1.0. Data are presented as mean ± SD, n = 3; ** p
    Figure Legend Snippet: Time- and dose-response relationships between oxLDL and the expression of Akt and p-Akt in wild-type EPC. a EPC cultures were exposed to nLDL (10 μg/ml) or oxLDL (1–50 μg/ml) in serum-free medium; the control well was treated with serum-free medium alone. Density of the Akt and p-Akt bands were first corrected with respect to β-actin (not shown) and then the ratio of p-Akt/total Akt expression calculated. The ratio of the wild-type group was assigned a value of 1.0. Data are presented as mean ± SD, n = 3; ** p

    Techniques Used: Expressing

    25) Product Images from "Hematein, a casein kinase II inhibitor, inhibits lung cancer tumor growth in a murine xenograft model"

    Article Title: Hematein, a casein kinase II inhibitor, inhibits lung cancer tumor growth in a murine xenograft model

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2013.2087

    Hematein inhibits cells growth, and inhibits Akt phosphorylation in A427 lung cancer cells. (A), A427 lung cancer cells were cultured in the absence and in increasing concentrations of hematein (10–100 μ M) as indicated. Cellular viability (normalized to DMSO control) was measured after 48 h using CellTiter-Glo ® Luminescent cell viability assay. Data points represent the average of IC50 value of hematein in triplet experiments and bars indicate SD. (B), After incubation with indicated concentrations of hematein for 2 weeks, colonies of A427 lung cancer cells were stained with 0.1% crystal violet, and colonies greater than 50 cells were counted. Results are expressed as relative colony formation: percentage of the number of colonies relative to the control group. Data represent the average of three independent experiments and bars indicate SEM. * p=0.0006, ** p=0.0001. (C), Phosphorylated Akt (Ser 129) was measured by western blot analysis. β-actin was used as an internal loading control. Band quantification was obtained by ImageJ software. Values are reported below each band and normalized to DMSO control.
    Figure Legend Snippet: Hematein inhibits cells growth, and inhibits Akt phosphorylation in A427 lung cancer cells. (A), A427 lung cancer cells were cultured in the absence and in increasing concentrations of hematein (10–100 μ M) as indicated. Cellular viability (normalized to DMSO control) was measured after 48 h using CellTiter-Glo ® Luminescent cell viability assay. Data points represent the average of IC50 value of hematein in triplet experiments and bars indicate SD. (B), After incubation with indicated concentrations of hematein for 2 weeks, colonies of A427 lung cancer cells were stained with 0.1% crystal violet, and colonies greater than 50 cells were counted. Results are expressed as relative colony formation: percentage of the number of colonies relative to the control group. Data represent the average of three independent experiments and bars indicate SEM. * p=0.0006, ** p=0.0001. (C), Phosphorylated Akt (Ser 129) was measured by western blot analysis. β-actin was used as an internal loading control. Band quantification was obtained by ImageJ software. Values are reported below each band and normalized to DMSO control.

    Techniques Used: Cell Culture, Cell Viability Assay, Incubation, Staining, Western Blot, Software

    26) Product Images from "Afzelin ameliorates D‐galactosamine and lipopolysaccharide‐induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics) Afzelin ameliorates D‐galactosamine and lipopolysaccharide‐induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics"

    Article Title: Afzelin ameliorates D‐galactosamine and lipopolysaccharide‐induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics) Afzelin ameliorates D‐galactosamine and lipopolysaccharide‐induced fulminant hepatic failure by modulating mitochondrial quality control and dynamics

    Journal: British Journal of Pharmacology

    doi: 10.1111/bph.13669

    Effect of mitophagy in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or CQ (60 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μgkg −1 ) treatment. Western blot analysis was performed to measure the protein levels of (A) hepatic ratio of LC3‐II/LC3‐I and (B) p62 in the CQ‐treated group. (C) Serum ALT, (D) mitochondrial GDH activity and (E) the rate of mitochondrial swelling were measured in the CQ‐treated group. The expression of each protein was adjusted to β‐actin as the loading control. The ratio of the LC3‐II and LC3‐I bands was evaluated by densitometric analysis. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P
    Figure Legend Snippet: Effect of mitophagy in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or CQ (60 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μgkg −1 ) treatment. Western blot analysis was performed to measure the protein levels of (A) hepatic ratio of LC3‐II/LC3‐I and (B) p62 in the CQ‐treated group. (C) Serum ALT, (D) mitochondrial GDH activity and (E) the rate of mitochondrial swelling were measured in the CQ‐treated group. The expression of each protein was adjusted to β‐actin as the loading control. The ratio of the LC3‐II and LC3‐I bands was evaluated by densitometric analysis. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P

    Techniques Used: Mouse Assay, Injection, Western Blot, Activity Assay, Expressing

    Effect of mitochondrial dynamics in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or mdivi‐1 (50 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. Western blot analysis was performed to measure the protein level of (A) hepatic Drp in the mdivi‐1‐treated group. (B) Serum ALT, (C) mitochondrial GDH activity and (D) the rate of mitochondrial swelling were measured in the mdivi‐1‐treated group. The expression of each protein was adjusted to β‐actin as the loading control. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P
    Figure Legend Snippet: Effect of mitochondrial dynamics in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or mdivi‐1 (50 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. Western blot analysis was performed to measure the protein level of (A) hepatic Drp in the mdivi‐1‐treated group. (B) Serum ALT, (C) mitochondrial GDH activity and (D) the rate of mitochondrial swelling were measured in the mdivi‐1‐treated group. The expression of each protein was adjusted to β‐actin as the loading control. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P

    Techniques Used: Mouse Assay, Injection, Western Blot, Activity Assay, Expressing

    Effect of afzelin on (A) hepatic Rev‐Erb‐α, (B) phospho‐AMPK and (C) SIRT1 protein expression in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or afzelin (200 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. The expression of each protein was adjusted to β‐actin as the loading control. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P
    Figure Legend Snippet: Effect of afzelin on (A) hepatic Rev‐Erb‐α, (B) phospho‐AMPK and (C) SIRT1 protein expression in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or afzelin (200 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. The expression of each protein was adjusted to β‐actin as the loading control. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P

    Techniques Used: Expressing, Mouse Assay, Injection

    Effect of afzelin on mitochondrial biogenesis and dynamics in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or afzelin (200 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. (A) mtDNA copy number was measured. Western blot analysis was performed to measure the protein levels of (B) hepatic PGC1α, (C) Nrf1, (D) TFAM (E) Drp1 and (F) Mfn2. The expression of each protein was adjusted to β‐actin as the loading control. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P
    Figure Legend Snippet: Effect of afzelin on mitochondrial biogenesis and dynamics in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or afzelin (200 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. (A) mtDNA copy number was measured. Western blot analysis was performed to measure the protein levels of (B) hepatic PGC1α, (C) Nrf1, (D) TFAM (E) Drp1 and (F) Mfn2. The expression of each protein was adjusted to β‐actin as the loading control. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P

    Techniques Used: Mouse Assay, Injection, Western Blot, Expressing

    Effect of afzelin on mitophagy in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or afzelin (200 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. Western blot analysis was performed to measure the protein levels of (A) mitochondrial PINK1 and (B) parkin as well as (C) the hepatic ratio of LC3‐II/LC3‐I and (D) p62 expression level in the afzelin‐treated group. The expression of each protein was adjusted to GAPDH or β‐actin as the loading control. The ratio of the LC3‐II and LC3‐I bands was evaluated by densitometric analysis. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P
    Figure Legend Snippet: Effect of afzelin on mitophagy in GalN/LPS‐treated mice. Mice received an i.p. injection of vehicle or afzelin (200 mg·kg −1 ) 1 h before GalN (800 mg·kg −1 )/LPS (40 μg·kg −1 ) treatment. Western blot analysis was performed to measure the protein levels of (A) mitochondrial PINK1 and (B) parkin as well as (C) the hepatic ratio of LC3‐II/LC3‐I and (D) p62 expression level in the afzelin‐treated group. The expression of each protein was adjusted to GAPDH or β‐actin as the loading control. The ratio of the LC3‐II and LC3‐I bands was evaluated by densitometric analysis. All values are expressed as mean ± SEM of 10 mice per group. * Significantly different ( P

    Techniques Used: Mouse Assay, Injection, Western Blot, Expressing

    27) Product Images from "FGFR gene alterations in lung squamous cell carcinoma are potential targets for the multikinase inhibitor nintedanib"

    Article Title: FGFR gene alterations in lung squamous cell carcinoma are potential targets for the multikinase inhibitor nintedanib

    Journal: Cancer Science

    doi: 10.1111/cas.13071

    Sensitivity of lung cancer cell lines positive for FGFR 1 copy number gain ( CNG ) to nintedanib. (a) FISH analysis of FGFR 1 copy number in lung cancer cell lines. The 5′ and 3′ probe signals for FGFR 1 appear green and red, respectively. Nuclei are stained blue with DAPI . (b) Immunoblot analysis of phosphorylated fibroblast growth factor receptor (p‐ FGFR ), FGFR 1, and β‐actin (loading control) in lung cancer cell lines positive (H520, H1581, LK ‐2) or negative (H1299, A549, PC ‐9) for FGFR 1 CNG . (c) Effects of nintedanib on the proliferation of lung cancer cell lines according to FGFR 1 copy number status. The IC 50 values are means ± SD from three independent experiments. (d) Effects of nintedanib on FGFR 1, ERK , and AKT phosphorylation in FGFR 1 CNG ‐positive lung squamous cell carcinoma cell lines. H520 and LK ‐2 cells were incubated for 6 h in the presence of the indicated concentrations of nintedanib, after which cell lysates (25 μg soluble protein) were subjected to immunoblot analysis with antibodies to the indicated proteins.
    Figure Legend Snippet: Sensitivity of lung cancer cell lines positive for FGFR 1 copy number gain ( CNG ) to nintedanib. (a) FISH analysis of FGFR 1 copy number in lung cancer cell lines. The 5′ and 3′ probe signals for FGFR 1 appear green and red, respectively. Nuclei are stained blue with DAPI . (b) Immunoblot analysis of phosphorylated fibroblast growth factor receptor (p‐ FGFR ), FGFR 1, and β‐actin (loading control) in lung cancer cell lines positive (H520, H1581, LK ‐2) or negative (H1299, A549, PC ‐9) for FGFR 1 CNG . (c) Effects of nintedanib on the proliferation of lung cancer cell lines according to FGFR 1 copy number status. The IC 50 values are means ± SD from three independent experiments. (d) Effects of nintedanib on FGFR 1, ERK , and AKT phosphorylation in FGFR 1 CNG ‐positive lung squamous cell carcinoma cell lines. H520 and LK ‐2 cells were incubated for 6 h in the presence of the indicated concentrations of nintedanib, after which cell lysates (25 μg soluble protein) were subjected to immunoblot analysis with antibodies to the indicated proteins.

    Techniques Used: Fluorescence In Situ Hybridization, Staining, Incubation

    28) Product Images from "Beneficial Effects of Metformin and/or Salicylate on Palmitate- or TNFα-Induced Neuroinflammatory Marker and Neuropeptide Gene Regulation in Immortalized NPY/AgRP Neurons"

    Article Title: Beneficial Effects of Metformin and/or Salicylate on Palmitate- or TNFα-Induced Neuroinflammatory Marker and Neuropeptide Gene Regulation in Immortalized NPY/AgRP Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0166973

    Phosphorylation of secondary-signaling molecules were regulated by metformin treatment in mHypoE-46 neurons. Cells were treated with 10, 20, 100 μM of metformin or H 2 O vehicle for 10 and 30 min. Protein was isolated for Western blot analysis. Levels of phospho-proteins were normalized to β-actin and expressed as mean ± SEM. Representative blots for phospho-proteins and β-actin are depicted beneath each graph. 20 μM metformin increased phosphorylation of AMPK (A) and S6K (B) at 10 min ( *p
    Figure Legend Snippet: Phosphorylation of secondary-signaling molecules were regulated by metformin treatment in mHypoE-46 neurons. Cells were treated with 10, 20, 100 μM of metformin or H 2 O vehicle for 10 and 30 min. Protein was isolated for Western blot analysis. Levels of phospho-proteins were normalized to β-actin and expressed as mean ± SEM. Representative blots for phospho-proteins and β-actin are depicted beneath each graph. 20 μM metformin increased phosphorylation of AMPK (A) and S6K (B) at 10 min ( *p

    Techniques Used: Isolation, Western Blot

    Phosphorylation of secondary-signaling molecules were regulated by sodium salicylate treatment in mHypoE-46 neurons. Cells were treated with 1 or 20 mM of sodium salicylate or H 2 O vehicle for 10 and 30 min. Protein was isolated for Western blot analysis. Levels of phospho-proteins were normalized to β-actin and expressed as mean ± SEM. Representative blots for phospho-proteins and β-actin are depicted beneath each graph. 20 mM sodium salicylate increased AMPK (A) phosphorylation and decreased S6K (B) phosphorylation at 10 ( *p
    Figure Legend Snippet: Phosphorylation of secondary-signaling molecules were regulated by sodium salicylate treatment in mHypoE-46 neurons. Cells were treated with 1 or 20 mM of sodium salicylate or H 2 O vehicle for 10 and 30 min. Protein was isolated for Western blot analysis. Levels of phospho-proteins were normalized to β-actin and expressed as mean ± SEM. Representative blots for phospho-proteins and β-actin are depicted beneath each graph. 20 mM sodium salicylate increased AMPK (A) phosphorylation and decreased S6K (B) phosphorylation at 10 ( *p

    Techniques Used: Isolation, Western Blot

    29) Product Images from "cJun NH2-terminal kinase mediates Interleukin-1? induced inhibition of lacrimal gland secretion."

    Article Title: cJun NH2-terminal kinase mediates Interleukin-1? induced inhibition of lacrimal gland secretion.

    Journal: Journal of neurochemistry

    doi: 10.1111/j.1471-4159.2005.03529.x

    Effect of SP600125 on IL-1 β induced JNK, p38, and Erk activation and phosphorylation of cJun. Lacrimal gland lobules were preincubated for 30 minutes in the absence or presence of increasing concentrations of SP600125. They were then incubated for 30 minutes in the absence (control, C) or presence of IL-1β (10 ng/ml). Proteins in the cell lysate were separated by SDS-PAGE followed by western blotting using antibodies against phosphorylated JNK or total JNK (A), phosphorylated cJun or total cJun (B), phosphorylated p38 or β-actin (C), phosphorylated ERK or p42ERK (D). Data in the plots are means ± SEM of three independent experiments. * Denotes statistically significant difference from control. # Denotes statistically significant difference from IL-1β. The blots shown in C and D are representative of data obtained in 2 other experiments.
    Figure Legend Snippet: Effect of SP600125 on IL-1 β induced JNK, p38, and Erk activation and phosphorylation of cJun. Lacrimal gland lobules were preincubated for 30 minutes in the absence or presence of increasing concentrations of SP600125. They were then incubated for 30 minutes in the absence (control, C) or presence of IL-1β (10 ng/ml). Proteins in the cell lysate were separated by SDS-PAGE followed by western blotting using antibodies against phosphorylated JNK or total JNK (A), phosphorylated cJun or total cJun (B), phosphorylated p38 or β-actin (C), phosphorylated ERK or p42ERK (D). Data in the plots are means ± SEM of three independent experiments. * Denotes statistically significant difference from control. # Denotes statistically significant difference from IL-1β. The blots shown in C and D are representative of data obtained in 2 other experiments.

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

    30) Product Images from "Histone deacetylases 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in acute myeloid leukemia cells"

    Article Title: Histone deacetylases 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in acute myeloid leukemia cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.14062

    FK228 decreases expression of BRCA1, CHK1, and RAD51 by inhibiting HDAC1 and HDAC2 in AML cells ( A and B ) THP-1 and OCI-AML3 cells were treated with FK228 for 48 h and whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( C ) THP-1 and OCI-AML3 cells were treated with FK228 for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analyses. **indicates p
    Figure Legend Snippet: FK228 decreases expression of BRCA1, CHK1, and RAD51 by inhibiting HDAC1 and HDAC2 in AML cells ( A and B ) THP-1 and OCI-AML3 cells were treated with FK228 for 48 h and whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( C ) THP-1 and OCI-AML3 cells were treated with FK228 for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analyses. **indicates p

    Techniques Used: Expressing, Western Blot, Staining, Flow Cytometry, Cytometry

    Inhibition of HDACs 1, 2, and 3 decreases the protein and transcript levels of BRCA1, CHK1, and RAD51, and induces apoptosis in AML cell lines ( A ) THP-1 cells were treated with variable concentrations of MGCD0103 for 48 h. Protein extracts were subjected to immunoprecipitation with antibodies against class I HDACs and then class I HDAC activities were measured, as described in the Materials and Methods. ( B ) THP-1 cells were treated with MGCD0103 for 48 h. Then total RNAs were isolated and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. ( C ) THP-1 cells were treated with MGCD0103 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( D ) OCI-AML3 cells were treated with MGCD0103 for 48 h, then total RNAs were isolated from treated cells and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. ( E ) OCI-AML3 cells were treated with MGCD0103 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. ( F ) THP-1 and OCI-AML3 cells were treated with MGCD0103 for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analysis. *indicates p
    Figure Legend Snippet: Inhibition of HDACs 1, 2, and 3 decreases the protein and transcript levels of BRCA1, CHK1, and RAD51, and induces apoptosis in AML cell lines ( A ) THP-1 cells were treated with variable concentrations of MGCD0103 for 48 h. Protein extracts were subjected to immunoprecipitation with antibodies against class I HDACs and then class I HDAC activities were measured, as described in the Materials and Methods. ( B ) THP-1 cells were treated with MGCD0103 for 48 h. Then total RNAs were isolated and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. ( C ) THP-1 cells were treated with MGCD0103 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. ( D ) OCI-AML3 cells were treated with MGCD0103 for 48 h, then total RNAs were isolated from treated cells and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. ( E ) OCI-AML3 cells were treated with MGCD0103 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. ( F ) THP-1 and OCI-AML3 cells were treated with MGCD0103 for 48 h and then subjected to Annexin V-FITC/PI staining and flow cytometry analysis. *indicates p

    Techniques Used: Inhibition, Immunoprecipitation, Isolation, Quantitative RT-PCR, Expressing, Western Blot, Staining, Flow Cytometry, Cytometry

    HDACs 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in AML cells ( A and C ) THP-1 cells were infected with HDAC1 (THP-1/HDAC1), HDAC2 (THP-1/HDAC2), HDAC3 (THP-1/HDAC3), or NTC control (THP-1/NTC) shRNA lentivirus overnight, then washed and incubated for 48 h prior to adding puromycin to the culture medium. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. These Western blots were previously published [ 15 ]. ( B ) Total RNAs were isolated from the transfected cells and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. ( D and E ) THP-1/HDAC1 and THP-1/HDAC2 cells were treated with RGFP966 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies.
    Figure Legend Snippet: HDACs 1 and 2 cooperate in regulating BRCA1, CHK1, and RAD51 expression in AML cells ( A and C ) THP-1 cells were infected with HDAC1 (THP-1/HDAC1), HDAC2 (THP-1/HDAC2), HDAC3 (THP-1/HDAC3), or NTC control (THP-1/NTC) shRNA lentivirus overnight, then washed and incubated for 48 h prior to adding puromycin to the culture medium. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies. The fold changes for the densitometry measurements, normalized to β-actin and then compared to no drug treatment control, are indicated. These Western blots were previously published [ 15 ]. ( B ) Total RNAs were isolated from the transfected cells and gene transcript levels were determined by Real-time RT-PCR. Transcript levels were normalized to GAPDH and relative expression levels were calculated using the comparative Ct method. ( D and E ) THP-1/HDAC1 and THP-1/HDAC2 cells were treated with RGFP966 for 48 h. Whole cell lysates were subjected to Western blotting and probed with the indicated antibodies.

    Techniques Used: Expressing, Infection, shRNA, Incubation, Western Blot, Isolation, Transfection, Quantitative RT-PCR

    31) Product Images from "Erythroid-Specific Expression of ?-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells"

    Article Title: Erythroid-Specific Expression of ?-globin from Sleeping Beauty-Transduced Human Hematopoietic Progenitor Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029110

    IHK-transduced CD34 + cells show β-globin expression only in erythroid cells. Thirty µg of total protein from IHK-transduced CD34 + cells differentiated into T-cells, B-cells, myeloid/monocytes, and granulocytes and 100 ng of total protein from non-transduced control or IHK-transduced CD34 + cells differentiated into erythrocytes were subjected to western blot using antibodies specific for β-globin and β-actin.
    Figure Legend Snippet: IHK-transduced CD34 + cells show β-globin expression only in erythroid cells. Thirty µg of total protein from IHK-transduced CD34 + cells differentiated into T-cells, B-cells, myeloid/monocytes, and granulocytes and 100 ng of total protein from non-transduced control or IHK-transduced CD34 + cells differentiated into erythrocytes were subjected to western blot using antibodies specific for β-globin and β-actin.

    Techniques Used: Expressing, Western Blot

    Increased β-globin transcript is detectable in non-erythroid progeny of IHK-transduced CD34 + cells. Twenty ng of total RNA was used for RT-PCR specific for spliced β-globin transcript and spliced β-actin. RNA from adult RBCs and erythrocyte-depleted adult WBCs was used as control.
    Figure Legend Snippet: Increased β-globin transcript is detectable in non-erythroid progeny of IHK-transduced CD34 + cells. Twenty ng of total RNA was used for RT-PCR specific for spliced β-globin transcript and spliced β-actin. RNA from adult RBCs and erythrocyte-depleted adult WBCs was used as control.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction

    Transduced CD34 + cells retain IHK transgene after expansion and differentiation. Total genomic DNA of 60 ng was PCR-amplified to detect the presence of IHK–β-globin, SB 100X, and β-actin control.
    Figure Legend Snippet: Transduced CD34 + cells retain IHK transgene after expansion and differentiation. Total genomic DNA of 60 ng was PCR-amplified to detect the presence of IHK–β-globin, SB 100X, and β-actin control.

    Techniques Used: Polymerase Chain Reaction, Amplification

    32) Product Images from "Analysis of MLN4924 (Pevonedistat) as a Potential Therapeutic Agent in Malignant Melanoma"

    Article Title: Analysis of MLN4924 (Pevonedistat) as a Potential Therapeutic Agent in Malignant Melanoma

    Journal: Melanoma research

    doi: 10.1097/CMR.0000000000000474

    MLN4924 treatment induces cleavage of apoptotic proteins A375 melanoma cells were treated for 72 hours with 0 nM, 1200 nM or 600 nM MLN4924. Cells were lysed and protein was harvested and subjected to immunoblot analysis for total or cleaved caspase-3, caspase-7, caspase-9 or PARP. β-actin was utilized as a loading control.
    Figure Legend Snippet: MLN4924 treatment induces cleavage of apoptotic proteins A375 melanoma cells were treated for 72 hours with 0 nM, 1200 nM or 600 nM MLN4924. Cells were lysed and protein was harvested and subjected to immunoblot analysis for total or cleaved caspase-3, caspase-7, caspase-9 or PARP. β-actin was utilized as a loading control.

    Techniques Used:

    33) Product Images from "The Effects of Combined Cyclic Stretch and Pressure on the Aortic Valve Interstitial Cell Phenotype"

    Article Title: The Effects of Combined Cyclic Stretch and Pressure on the Aortic Valve Interstitial Cell Phenotype

    Journal: Annals of biomedical engineering

    doi: 10.1007/s10439-011-0273-x

    (a) Immunoblot bands for Caldesmon, α -SMA, and β -actin (b, c, d) expression of the various proteins normalized to β -actin and then the 10% stretch case (* p
    Figure Legend Snippet: (a) Immunoblot bands for Caldesmon, α -SMA, and β -actin (b, c, d) expression of the various proteins normalized to β -actin and then the 10% stretch case (* p

    Techniques Used: Expressing

    34) Product Images from "Protective Effects of Casticin From Vitex trifolia Alleviate Eosinophilic Airway Inflammation and Oxidative Stress in a Murine Asthma Model"

    Article Title: Protective Effects of Casticin From Vitex trifolia Alleviate Eosinophilic Airway Inflammation and Oxidative Stress in a Murine Asthma Model

    Journal: Frontiers in Pharmacology

    doi: 10.3389/fphar.2018.00635

    Effects of casticin (CAS) on the expression of chemokines, inflammatory mediators, and cytokines in the lungs. Gene expression was determined using real-time RT-PCR with lung tissues from normal (N) and OVA-stimulated (OVA) mice with or without CAS and prednisolone (P) treatment. Fold-changes in expression were measured relative to β-actin expression (internal control). The data are presented as means ± SEM of three independent experiments ( n = 8). ∗ p
    Figure Legend Snippet: Effects of casticin (CAS) on the expression of chemokines, inflammatory mediators, and cytokines in the lungs. Gene expression was determined using real-time RT-PCR with lung tissues from normal (N) and OVA-stimulated (OVA) mice with or without CAS and prednisolone (P) treatment. Fold-changes in expression were measured relative to β-actin expression (internal control). The data are presented as means ± SEM of three independent experiments ( n = 8). ∗ p

    Techniques Used: Expressing, Quantitative RT-PCR, Mouse Assay

    35) Product Images from "Heme oxygenase-1-transduced bone marrow mesenchymal stem cells in reducing acute rejection and improving small bowel transplantation outcomes in rats"

    Article Title: Heme oxygenase-1-transduced bone marrow mesenchymal stem cells in reducing acute rejection and improving small bowel transplantation outcomes in rats

    Journal: Stem Cell Research & Therapy

    doi: 10.1186/s13287-016-0427-8

    Expression of HO-1 protein and mRNA in the graft after SBTx. Expression of HO-1 in protein ( a ) and mRNA ( b ) was confirmed by western blot assay and real-time PCR, respectively ( n = 5 in each group). Relative content was determined by HO-1/β-actin ratio. High-level and steady expression of HO-1 protein and mRNA were observed in the Ad/HO-1/BMMSC group (* P
    Figure Legend Snippet: Expression of HO-1 protein and mRNA in the graft after SBTx. Expression of HO-1 in protein ( a ) and mRNA ( b ) was confirmed by western blot assay and real-time PCR, respectively ( n = 5 in each group). Relative content was determined by HO-1/β-actin ratio. High-level and steady expression of HO-1 protein and mRNA were observed in the Ad/HO-1/BMMSC group (* P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction

    36) Product Images from "Co-delivery of Doxorubicin and Bmi1 siRNA by Folate Receptor Targeted Liposomes Exhibits Enhanced Anti-Tumor Effects in vitro and in vivo"

    Article Title: Co-delivery of Doxorubicin and Bmi1 siRNA by Folate Receptor Targeted Liposomes Exhibits Enhanced Anti-Tumor Effects in vitro and in vivo

    Journal: Theranostics

    doi: 10.7150/thno.9423

    The Bmi1 siRNA delivery efficiency by FA-siRNA-L and repression effects on Bmi1 expression in KB cells. For western blotting and qRT-PCR assay, cells cultured in DMEM medium were harvested and protein or mRNA was extracted for the determinations. For the uptake study, KB and LO2 cells grown in a monolayer were incubated with FA-siRNA-L, siRNA-L, and Chol-siRNA for 1 h at 37°C and were then used for the next assays. A. The Bmi1 protein expression in cancer or normal cell lines detected by western blot. B. QRT-PCR quantitative analysis of the expression of Bmi1 in cancer cells in mRNA level. C. Cellular uptake and intracellular distribution of Bmi1 siRNA in the KB cells. D. Western blotting of Bmi1 expression in the cells treated with Bmi1 siRNA complex or liposomes. LO2 cells were used as the normal cell control and β-actin was used as the loading control. E. Quantitative analysis of the western blotting bands. Data are expressed as mean ± SD of 3 independent samples. **: P
    Figure Legend Snippet: The Bmi1 siRNA delivery efficiency by FA-siRNA-L and repression effects on Bmi1 expression in KB cells. For western blotting and qRT-PCR assay, cells cultured in DMEM medium were harvested and protein or mRNA was extracted for the determinations. For the uptake study, KB and LO2 cells grown in a monolayer were incubated with FA-siRNA-L, siRNA-L, and Chol-siRNA for 1 h at 37°C and were then used for the next assays. A. The Bmi1 protein expression in cancer or normal cell lines detected by western blot. B. QRT-PCR quantitative analysis of the expression of Bmi1 in cancer cells in mRNA level. C. Cellular uptake and intracellular distribution of Bmi1 siRNA in the KB cells. D. Western blotting of Bmi1 expression in the cells treated with Bmi1 siRNA complex or liposomes. LO2 cells were used as the normal cell control and β-actin was used as the loading control. E. Quantitative analysis of the western blotting bands. Data are expressed as mean ± SD of 3 independent samples. **: P

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Cell Culture, Incubation

    37) Product Images from "Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer"

    Article Title: Endoplasmic reticulum stress induces PRNP prion protein gene expression in breast cancer

    Journal: Breast Cancer Research : BCR

    doi: 10.1186/bcr3398

    PrP delays cell death induced by ER stress . (A) inset shows western blot of PrP and β-actin in siRNA transfected cells. Percentage of cell death assessed by pan-caspase FLICA (A) or by chromatin condensation (B) in MCF-7 cells transfected with control (siCtl) or PrP (siPrP) siRNAs for 24 hrs and treated with ER stressors or DMSO (control) during 6 hrs. Cell death was measured 18 hrs after the removal of ER stress. Data represent the mean ± SEM of three (A) or six (B) independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP siRNA and control siRNA. (C-D) Percentage of cell death assessed by FLICA staining for active caspases (C) or by chromatin condensation in PrP +/+ and PrP-/- hippocampal cell lines (D). Data represent the mean ± SEM of four independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP+/+ and PrP-/- cells. C Inset: Western blot analysis of PrP with the R155 antibody and β-actin in protein extracts from mouse PrP+/+ and PrP-/- hippocampal cell lines. PrP-/- hippocampal cell lines were treated with 2.5 μg/ml of ER stressors. These concentrations were empirically determined to induce ER stress response without initially inducing strong toxic effects. (E) Relative fluorescence units (RFU) of FLICA activity representing pan-caspase activity in MDA-MB-231 (E) or HS578T (F) cells transfected with siCtl or siPrP.
    Figure Legend Snippet: PrP delays cell death induced by ER stress . (A) inset shows western blot of PrP and β-actin in siRNA transfected cells. Percentage of cell death assessed by pan-caspase FLICA (A) or by chromatin condensation (B) in MCF-7 cells transfected with control (siCtl) or PrP (siPrP) siRNAs for 24 hrs and treated with ER stressors or DMSO (control) during 6 hrs. Cell death was measured 18 hrs after the removal of ER stress. Data represent the mean ± SEM of three (A) or six (B) independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP siRNA and control siRNA. (C-D) Percentage of cell death assessed by FLICA staining for active caspases (C) or by chromatin condensation in PrP +/+ and PrP-/- hippocampal cell lines (D). Data represent the mean ± SEM of four independent experiments. At least 150 cells were counted per experiment. * Indicates P ≤0.05 between PrP+/+ and PrP-/- cells. C Inset: Western blot analysis of PrP with the R155 antibody and β-actin in protein extracts from mouse PrP+/+ and PrP-/- hippocampal cell lines. PrP-/- hippocampal cell lines were treated with 2.5 μg/ml of ER stressors. These concentrations were empirically determined to induce ER stress response without initially inducing strong toxic effects. (E) Relative fluorescence units (RFU) of FLICA activity representing pan-caspase activity in MDA-MB-231 (E) or HS578T (F) cells transfected with siCtl or siPrP.

    Techniques Used: Western Blot, Transfection, Staining, Fluorescence, Activity Assay, Multiple Displacement Amplification

    PrP does not prevent ER stress-mediated Bax activation in MCF-7 cells . Western blot assessing the impact of PrP silencing on Bax activation as well as Bim and Bcl-2 levels at 6, 12 or 18 h following a 6-h treatment with Brefeldin A (A) , Tunicamycin (B) , or Thapsigar gin (C) . Western blot using the 3F4 antibody confirms ER stress-induced increase in PrP levels as well as its efficient siRNA-mediated silencing, while probing for Bax and β-actin controls for equal protein input and protein loading, respectively. ( D) MCF-7 cells transfected with a second siPrP and treated for 18 h with ER stressor; experiments performed as described in A-C.
    Figure Legend Snippet: PrP does not prevent ER stress-mediated Bax activation in MCF-7 cells . Western blot assessing the impact of PrP silencing on Bax activation as well as Bim and Bcl-2 levels at 6, 12 or 18 h following a 6-h treatment with Brefeldin A (A) , Tunicamycin (B) , or Thapsigar gin (C) . Western blot using the 3F4 antibody confirms ER stress-induced increase in PrP levels as well as its efficient siRNA-mediated silencing, while probing for Bax and β-actin controls for equal protein input and protein loading, respectively. ( D) MCF-7 cells transfected with a second siPrP and treated for 18 h with ER stressor; experiments performed as described in A-C.

    Techniques Used: Activation Assay, Western Blot, Transfection

    ER stress transcriptionally increases PrP levels in MCF-7 cells . One representative Western blot from at least three independent experiments is shown in A-B . (A) Western blots of PrP with the 3F4 antibody, BiP, and β-actin in protein extracts from MCF-7 cells treated 18 hrs with increasing concentrations of Brefeldin A, Tunicamycin, or Thapsigargin. (B) Western blot of PrP (3F4) and β-actin in protein extracts from MCF-7 cells incubated for 18 hrs in the absence or presence of serum or transfected with pCep4β-PrP (pC-PrP). (C) PrP, XBP1, sXBP1 and β-actin RT-PCR cDNA amplicons from cells treated for 6 hrs. The ratio of PrP over β-actin was calculated from three independent experiments. ( D ) Western blot of various ER stress-regulated proteins in MCF-7 cells treated 18 hrs with BFA, TM or Thps. (E) Western blot of PrP with the 3F4 PrP, CHOP and β-actin antibodies in protein extracts from MCF-7 cells treated 18 hrs with DMSO (Ctl) or ER stressors in the presence or in absence of cycloheximide (CHX) or actinomycin D (Act D). The immunoreactive band at 37 kDa was not consistently detected with the anti-PrP 3F4 antibody suggesting a non-specific band (ns).
    Figure Legend Snippet: ER stress transcriptionally increases PrP levels in MCF-7 cells . One representative Western blot from at least three independent experiments is shown in A-B . (A) Western blots of PrP with the 3F4 antibody, BiP, and β-actin in protein extracts from MCF-7 cells treated 18 hrs with increasing concentrations of Brefeldin A, Tunicamycin, or Thapsigargin. (B) Western blot of PrP (3F4) and β-actin in protein extracts from MCF-7 cells incubated for 18 hrs in the absence or presence of serum or transfected with pCep4β-PrP (pC-PrP). (C) PrP, XBP1, sXBP1 and β-actin RT-PCR cDNA amplicons from cells treated for 6 hrs. The ratio of PrP over β-actin was calculated from three independent experiments. ( D ) Western blot of various ER stress-regulated proteins in MCF-7 cells treated 18 hrs with BFA, TM or Thps. (E) Western blot of PrP with the 3F4 PrP, CHOP and β-actin antibodies in protein extracts from MCF-7 cells treated 18 hrs with DMSO (Ctl) or ER stressors in the presence or in absence of cycloheximide (CHX) or actinomycin D (Act D). The immunoreactive band at 37 kDa was not consistently detected with the anti-PrP 3F4 antibody suggesting a non-specific band (ns).

    Techniques Used: Western Blot, Incubation, Transfection, Reverse Transcription Polymerase Chain Reaction, CTL Assay, Activated Clotting Time Assay

    ER stress increases PrP levels in MDA-MB-231, HS578T and HCC1500 cell lines . Western blot of ( A) PrP in protein extracts of MCF-7, MDA-MB-231, HS578T and HCC1500 cell lines, ( B) PrP and BiP in protein extracts from HCC1500 cells treated with BFA, TM or Thps, ( C ) PrP and BiP in protein extracts from MCF-7, MDA-MB-231 and HS578T cell lines treated with 4-PBA or vehicle, ( D ) PrP and BiP in protein extracts of MDA-MB-231 and HS578T cell lines treated with BFA, TM and Thps. β-actin was probed as a loading control.
    Figure Legend Snippet: ER stress increases PrP levels in MDA-MB-231, HS578T and HCC1500 cell lines . Western blot of ( A) PrP in protein extracts of MCF-7, MDA-MB-231, HS578T and HCC1500 cell lines, ( B) PrP and BiP in protein extracts from HCC1500 cells treated with BFA, TM or Thps, ( C ) PrP and BiP in protein extracts from MCF-7, MDA-MB-231 and HS578T cell lines treated with 4-PBA or vehicle, ( D ) PrP and BiP in protein extracts of MDA-MB-231 and HS578T cell lines treated with BFA, TM and Thps. β-actin was probed as a loading control.

    Techniques Used: Multiple Displacement Amplification, Western Blot

    sXBP1 is involved in PRNP gene expression in MCF-7 and MDA-MB-231 cells . (A) Western blot analyses of PrP with the 3F4 antibody, cleaved ATF6α (ΔATF6α), phosphorylated eIF2α (peIF2α), total eIF2α, and β-actin. Lower panels represent XBP1, spliced XBP1 (sXBP1) and β-actin amplified by RT-PCR. Protein or mRNA extracts were from MCF-7 cells treated with ER stressors for 0 to 6 hrs. The increase of PrP and ΔATF6α levels compared to β-actin levels and the ratios of peIF2α/eIF2α and sXBP1/XBP1 are indicated. (B) MCF-7 cells transfected with siATF6α or siXBP1 and proteins immunoblotted with 3F4 PrP and β-actin antibodies. RT-PCR shows levels of ATF6α and XBP1 mRNAs. (C) Western blot analyses of PrP, HA tag and β-actin in protein extracts from MCF-7 cells transfected for 6 hrs with pCGN-IRES-EGFP (Ctl), pCGN-HA-sXBP1-IRES-EGFP, and pCGN-HA-ATF4-IRES-EGFP constructs. (D) ChIP assays performed on DMSO (Ctl)- or BFA-treated MCF-7 cells with IgG control, XBP1 or ATF6α antibodies. PCR amplification of PRNP and β-actin gene promoters ( ACTB ) was done on immunoprecipitated and non-immunoprecipitated (input) DNA. ( E ) Western blot of PrP (top panel) and β-actin (bottom panel) and ethidium stained agarose gel containing ATF6α and XBP1 amplicons from MDA-MB-231 or HS578T cells transfected with siCtl, siATF6α or siXBP1. NT indicates non-transfected, D indicates the Dharmacon siRNAs and SC indicates the Santa Cruz siRNAs. ( F) Levels of PRNP mRNA detected by qRT-PCR in siATF6α or siXBP1-transfected cells.
    Figure Legend Snippet: sXBP1 is involved in PRNP gene expression in MCF-7 and MDA-MB-231 cells . (A) Western blot analyses of PrP with the 3F4 antibody, cleaved ATF6α (ΔATF6α), phosphorylated eIF2α (peIF2α), total eIF2α, and β-actin. Lower panels represent XBP1, spliced XBP1 (sXBP1) and β-actin amplified by RT-PCR. Protein or mRNA extracts were from MCF-7 cells treated with ER stressors for 0 to 6 hrs. The increase of PrP and ΔATF6α levels compared to β-actin levels and the ratios of peIF2α/eIF2α and sXBP1/XBP1 are indicated. (B) MCF-7 cells transfected with siATF6α or siXBP1 and proteins immunoblotted with 3F4 PrP and β-actin antibodies. RT-PCR shows levels of ATF6α and XBP1 mRNAs. (C) Western blot analyses of PrP, HA tag and β-actin in protein extracts from MCF-7 cells transfected for 6 hrs with pCGN-IRES-EGFP (Ctl), pCGN-HA-sXBP1-IRES-EGFP, and pCGN-HA-ATF4-IRES-EGFP constructs. (D) ChIP assays performed on DMSO (Ctl)- or BFA-treated MCF-7 cells with IgG control, XBP1 or ATF6α antibodies. PCR amplification of PRNP and β-actin gene promoters ( ACTB ) was done on immunoprecipitated and non-immunoprecipitated (input) DNA. ( E ) Western blot of PrP (top panel) and β-actin (bottom panel) and ethidium stained agarose gel containing ATF6α and XBP1 amplicons from MDA-MB-231 or HS578T cells transfected with siCtl, siATF6α or siXBP1. NT indicates non-transfected, D indicates the Dharmacon siRNAs and SC indicates the Santa Cruz siRNAs. ( F) Levels of PRNP mRNA detected by qRT-PCR in siATF6α or siXBP1-transfected cells.

    Techniques Used: Expressing, Multiple Displacement Amplification, Western Blot, Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection, CTL Assay, Construct, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Immunoprecipitation, Staining, Agarose Gel Electrophoresis, Quantitative RT-PCR

    38) Product Images from "Neuroprotective Effects of a Smoothened Receptor Agonist against Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats"

    Article Title: Neuroprotective Effects of a Smoothened Receptor Agonist against Early Brain Injury after Experimental Subarachnoid Hemorrhage in Rats

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2016.00306

    Effects of treatment with PUR on Bax, Bcl-2 in mRNA and protein levels. (A) The relative expression levels of Bax and Bcl-2 mRNA in the PFC were analyzed by RT-PCR. The densities of the protein bands were analyzed and normalized to β-actin, n = 3. (B) Representative western blots showing levels of Bax and Bcl-2 in the PFC, Bar graphs showing quantification of the protein levels of Bax and Bcl-2, n = 3. Both the determination of the two signs in mRNA and protein levels were obtained from three separate experiments. Values represent the mean ± SD. *** p
    Figure Legend Snippet: Effects of treatment with PUR on Bax, Bcl-2 in mRNA and protein levels. (A) The relative expression levels of Bax and Bcl-2 mRNA in the PFC were analyzed by RT-PCR. The densities of the protein bands were analyzed and normalized to β-actin, n = 3. (B) Representative western blots showing levels of Bax and Bcl-2 in the PFC, Bar graphs showing quantification of the protein levels of Bax and Bcl-2, n = 3. Both the determination of the two signs in mRNA and protein levels were obtained from three separate experiments. Values represent the mean ± SD. *** p

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Effects of PUR on Shh pathway . The quantification of Shh, Gli-1, and Ptch was measured by RT-PCR (A) and western blot (B) 48 h post-SAH. Each value was normalized to β-actin. Bar graphs showing quantification of mRNA and protein levels of Shh, Gli-1, and Ptch were determined by Image-Pro Plus 6.0, n = 3. Values represent the mean ± SD. ** p
    Figure Legend Snippet: Effects of PUR on Shh pathway . The quantification of Shh, Gli-1, and Ptch was measured by RT-PCR (A) and western blot (B) 48 h post-SAH. Each value was normalized to β-actin. Bar graphs showing quantification of mRNA and protein levels of Shh, Gli-1, and Ptch were determined by Image-Pro Plus 6.0, n = 3. Values represent the mean ± SD. ** p

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Western Blot

    39) Product Images from "C/EBP? triggers proteasome-dependent degradation of cdk4 during growth arrest"

    Article Title: C/EBP? triggers proteasome-dependent degradation of cdk4 during growth arrest

    Journal: The EMBO Journal

    doi: 10.1093/emboj/21.5.930

    Fig. 2. The half-life of cdk4 is increased in primary hepatocytes lacking C/EBPα. ( A ) Protein levels of cdk4 are induced in C/EBPα-knockout primary hepatocytes. Nuclear extracts from wild-type (+/+) and C/EBPα-knockout (–/–) hepatocytes were analyzed by western blotting with antibodies to cdk4. cdk4 levels were calculated as the ratio to β-actin and are shown below. ( B ) Induction of cdk4 levels in C/EBPα-knockout animals. The cdk4:β-actin ratio was calculated in wild-type and C/EBPα-knockout primary hepatocytes. A summary of three experiments is shown. ( C ) The half-life of cdk4 in wild-type and C/EBPα-knockout primary hepatocytes. The inhibitor of protein synthesis, CHX (10 µg/ml), was added to the cells. Proteins were then isolated at different time points after CHX addition (shown on the top) and analyzed by western blotting with antibodies to cdk4. The levels of cdk4 (expressed as a ratio to β-actin) are shown below. ( D ) The proteasome inhibitor MG132 blocks the C/EBPα-dependent degradation of cdk4. MG132 (50 µM) was added to the cells 1 h prior to CHX addition and the experiment was performed as described above.
    Figure Legend Snippet: Fig. 2. The half-life of cdk4 is increased in primary hepatocytes lacking C/EBPα. ( A ) Protein levels of cdk4 are induced in C/EBPα-knockout primary hepatocytes. Nuclear extracts from wild-type (+/+) and C/EBPα-knockout (–/–) hepatocytes were analyzed by western blotting with antibodies to cdk4. cdk4 levels were calculated as the ratio to β-actin and are shown below. ( B ) Induction of cdk4 levels in C/EBPα-knockout animals. The cdk4:β-actin ratio was calculated in wild-type and C/EBPα-knockout primary hepatocytes. A summary of three experiments is shown. ( C ) The half-life of cdk4 in wild-type and C/EBPα-knockout primary hepatocytes. The inhibitor of protein synthesis, CHX (10 µg/ml), was added to the cells. Proteins were then isolated at different time points after CHX addition (shown on the top) and analyzed by western blotting with antibodies to cdk4. The levels of cdk4 (expressed as a ratio to β-actin) are shown below. ( D ) The proteasome inhibitor MG132 blocks the C/EBPα-dependent degradation of cdk4. MG132 (50 µM) was added to the cells 1 h prior to CHX addition and the experiment was performed as described above.

    Techniques Used: Knock-Out, Western Blot, Isolation

    Fig. 1. Protein levels of cdk4 are induced in tissues of C/EBPα-knockout mice. ( A ) Western analysis of cdk4 expression in liver. Nuclear extracts or cytoplasm from newborn wild-type (WT), heterozygous (H) or C/EBPα-knockout (KO) livers were loaded on a denaturing gel and probed with antibodies to cdk4. The membranes were stripped and re-probed with antibodies to β-actin to verify protein loading. C, control extract containing high levels of cdk4. Protein levels of cdk4 were calculated as their ratio to β-actin. Summaries of three to five experiments are shown below as bar graphs. ( B ) Northern analysis of cdk4 mRNA in livers from wild-type, heterozygous and C/EBPα-knockout littermates. Results from two animals of each genotype are shown. The membrane was re-probed with 18S rRNA probe. The intensity of signals was determined using phosphoimaging and the levels of cdk4 mRNA were calculated as a ratio to 18S rRNA. The bar graph show a summary of data with five animals per genotype. ( C ).
    Figure Legend Snippet: Fig. 1. Protein levels of cdk4 are induced in tissues of C/EBPα-knockout mice. ( A ) Western analysis of cdk4 expression in liver. Nuclear extracts or cytoplasm from newborn wild-type (WT), heterozygous (H) or C/EBPα-knockout (KO) livers were loaded on a denaturing gel and probed with antibodies to cdk4. The membranes were stripped and re-probed with antibodies to β-actin to verify protein loading. C, control extract containing high levels of cdk4. Protein levels of cdk4 were calculated as their ratio to β-actin. Summaries of three to five experiments are shown below as bar graphs. ( B ) Northern analysis of cdk4 mRNA in livers from wild-type, heterozygous and C/EBPα-knockout littermates. Results from two animals of each genotype are shown. The membrane was re-probed with 18S rRNA probe. The intensity of signals was determined using phosphoimaging and the levels of cdk4 mRNA were calculated as a ratio to 18S rRNA. The bar graph show a summary of data with five animals per genotype. ( C ).

    Techniques Used: Knock-Out, Mouse Assay, Western Blot, Expressing, Northern Blot

    Fig. 6. C/EBPα reduces cdk4–cdc37–Hsp90 complex, induces cdk4–ubiquitin conjugates and shortens the half-life of cdk4 during growth arrest in HT1 cells. ( A ) Expression of HA-ubiquitin leads to the formation of cdk4–ubiquitin intermediates. COS7 cells were transfected with the empty vector (V, control) or with a plasmid expressing HA-ubiquitin. The next day after transfection, cells were treated with inhibitors of the proteasome MG132 or LlnL (L) or with DMSO (control). Cdk4 was precipitated, and cdk4 immunoprecipitations were analyzed by western blotting with antibodies to the HA-tag (upper) or with antibodies to cdk4 (lower). ( B ) cdk4 levels are reduced in cells growth arrested by C/EBPα. HT1 cells were synchronized by serum starvation, expression of C/EBPα was induced by IPTG, and cells were plated at low density. Levels of cdk4, cyclin A, cyclin D1 and C/EBPα were determined 30 h after plating in glucose-treated (control) and IPTG-treated cells (Western). β-actin shows the re-probe of the membrane for cdk4. BrdU uptake shows percentage of glucose- and IPTG-treated cells incorporating BrdU at 18 h after plating. ( C ) cdk4–ubiquitin conjugates are increased in cells arrested by C/EBPα. HT1 cells were synchronized in G 1 . C/EBPα was induced by IPTG and cells were plated at low density. Cdk4 was precipitated from cells at 2 and 30 h, and cdk4 immunoprecipitations were analyzed by western blotting with antibodies to ubiquitin, cdk4 and C/EBPα. The result shown in the figure represents the re-probe of the same membrane with the aforementioned antibodies. The position of the cdk4–ubiquitin conjugate induced by C/EBPα is shown on the right. ( D ) The half-life of cdk4 is reduced in growth-arrested HT1 cells. Protein synthesis was blocked by cyclohexamide (CHX) and cdk4 levels were examined by western blotting at 0, 2, 4 and 8 h after CHX addition. The stability of cdk4 was calculated as a ratio to 0 point. A summary of three experiments is shown below. ( E ) cdk4–cdc37–Hsp90 complex is reduced in C/EBPα growth-arrest cells. Total proteins from control cells (glucose) and from cells arrested by C/EBPα (IPTG) were fractionated by gel filtration, and Hsp90, cdc37 and cdk4 were examined by western blotting with specific antibodies.
    Figure Legend Snippet: Fig. 6. C/EBPα reduces cdk4–cdc37–Hsp90 complex, induces cdk4–ubiquitin conjugates and shortens the half-life of cdk4 during growth arrest in HT1 cells. ( A ) Expression of HA-ubiquitin leads to the formation of cdk4–ubiquitin intermediates. COS7 cells were transfected with the empty vector (V, control) or with a plasmid expressing HA-ubiquitin. The next day after transfection, cells were treated with inhibitors of the proteasome MG132 or LlnL (L) or with DMSO (control). Cdk4 was precipitated, and cdk4 immunoprecipitations were analyzed by western blotting with antibodies to the HA-tag (upper) or with antibodies to cdk4 (lower). ( B ) cdk4 levels are reduced in cells growth arrested by C/EBPα. HT1 cells were synchronized by serum starvation, expression of C/EBPα was induced by IPTG, and cells were plated at low density. Levels of cdk4, cyclin A, cyclin D1 and C/EBPα were determined 30 h after plating in glucose-treated (control) and IPTG-treated cells (Western). β-actin shows the re-probe of the membrane for cdk4. BrdU uptake shows percentage of glucose- and IPTG-treated cells incorporating BrdU at 18 h after plating. ( C ) cdk4–ubiquitin conjugates are increased in cells arrested by C/EBPα. HT1 cells were synchronized in G 1 . C/EBPα was induced by IPTG and cells were plated at low density. Cdk4 was precipitated from cells at 2 and 30 h, and cdk4 immunoprecipitations were analyzed by western blotting with antibodies to ubiquitin, cdk4 and C/EBPα. The result shown in the figure represents the re-probe of the same membrane with the aforementioned antibodies. The position of the cdk4–ubiquitin conjugate induced by C/EBPα is shown on the right. ( D ) The half-life of cdk4 is reduced in growth-arrested HT1 cells. Protein synthesis was blocked by cyclohexamide (CHX) and cdk4 levels were examined by western blotting at 0, 2, 4 and 8 h after CHX addition. The stability of cdk4 was calculated as a ratio to 0 point. A summary of three experiments is shown below. ( E ) cdk4–cdc37–Hsp90 complex is reduced in C/EBPα growth-arrest cells. Total proteins from control cells (glucose) and from cells arrested by C/EBPα (IPTG) were fractionated by gel filtration, and Hsp90, cdc37 and cdk4 were examined by western blotting with specific antibodies.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Western Blot, Filtration

    Fig. 3. ( A ) Expression of C/EBPα is increased at later stages of prenatal development. Nuclear extracts (200 µg) isolated from livers at 16, 18 and 20 (N, newborn) days of gestation were analyzed by western blotting with antibodies to C/EBPα. A pool of three livers from each genotype was used for the protein isolation. The membrane was re-probed with β-actin antibodies. Levels of C/EBPα were calculated as a ratio of C/EBPα (42 kDa + 30 kDa) to β-actin. CRM; cross reactive molecule. ( B ) CDK4 protein levels during prenatal development in wild-type and C/EBPα-knockout livers. Western blotting was performed with liver nuclear extracts from wild-type, heterozygous and C/EBPα-knockout mice isolated at different stages of prenatal development and from newborn mice (N). Livers from three animals per genotype were used for protein isolation. The same membrane was re-probed with antibodies to cdk4, cdk2, PCNA, cdc2 and β-actin. The levels of each protein were calculated as ratios to β-actin and are shown below each panel. Asterisk shows the position of a high molecular weight cdk4 immunoreactive protein. ( C ) High molecular weight cdk4 immunoreactive proteins are cdk4–ubiquitin conjugates. Ubiquitin (right) or cdk4 (left) were precipitated from nuclear extracts of wild-type animals with specific antibodies (monoclonal for ubiquitin, polyclonal for cdk4). Immunoprecipitations were analyzed by western blotting with monoclonal antibodies to cdk4 (right) or polyclonal antibodies to ubiquitin (left). Nuclear extracts from wild-type livers were run on the same gel with ubiquitin immuno precipitations and analyzed by western blotting with antibodies to cdk4. Positions of molecular weight markers are shown.
    Figure Legend Snippet: Fig. 3. ( A ) Expression of C/EBPα is increased at later stages of prenatal development. Nuclear extracts (200 µg) isolated from livers at 16, 18 and 20 (N, newborn) days of gestation were analyzed by western blotting with antibodies to C/EBPα. A pool of three livers from each genotype was used for the protein isolation. The membrane was re-probed with β-actin antibodies. Levels of C/EBPα were calculated as a ratio of C/EBPα (42 kDa + 30 kDa) to β-actin. CRM; cross reactive molecule. ( B ) CDK4 protein levels during prenatal development in wild-type and C/EBPα-knockout livers. Western blotting was performed with liver nuclear extracts from wild-type, heterozygous and C/EBPα-knockout mice isolated at different stages of prenatal development and from newborn mice (N). Livers from three animals per genotype were used for protein isolation. The same membrane was re-probed with antibodies to cdk4, cdk2, PCNA, cdc2 and β-actin. The levels of each protein were calculated as ratios to β-actin and are shown below each panel. Asterisk shows the position of a high molecular weight cdk4 immunoreactive protein. ( C ) High molecular weight cdk4 immunoreactive proteins are cdk4–ubiquitin conjugates. Ubiquitin (right) or cdk4 (left) were precipitated from nuclear extracts of wild-type animals with specific antibodies (monoclonal for ubiquitin, polyclonal for cdk4). Immunoprecipitations were analyzed by western blotting with monoclonal antibodies to cdk4 (right) or polyclonal antibodies to ubiquitin (left). Nuclear extracts from wild-type livers were run on the same gel with ubiquitin immuno precipitations and analyzed by western blotting with antibodies to cdk4. Positions of molecular weight markers are shown.

    Techniques Used: Expressing, Isolation, Western Blot, Knock-Out, Mouse Assay, Molecular Weight

    40) Product Images from "Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs"

    Article Title: Role played by Prx1-dependent extracellular matrix properties in vascular smooth muscle development in embryonic lungs

    Journal: Pulmonary Circulation

    doi: 10.1086/681272

    Prx1 regulates transforming growth factor (TGF)–β signaling in embryonic lungs. A , Immunohistochemical staining for TGF-β1 and TGF-β2 in lung sections derived from E15.5 ( top ) and D0 ( bottom ) Prx1 WT ( left ) and Prx1 null ( right ) mice. Brown indicates positive staining of antibodies. While TGF-β localization is restricted to the developing vessel area (arrows) in Prx1 WT lungs, in Prx1 null lungs TGF-β expression is observed in areas other than the vessel wall vicinity (arrowheads). Hematoxylin staining (blue) is used for nuclear counterstaining. Scale bar = 40 μm. B , Western blotting for TGF-β1 protein expression in E17.5 lungs from Prx1 WT and Prx1 null mice. C , Real-time polymerase chain reaction (PCR) for TGF-β1 messenger RNA (mRNA) expression in E17.5 lungs from Prx1 WT and Prx1 null mice. D , Western blotting for phosphorylated Smad2 (P-Smad2; top ) and total Smad 2/3 ( middle ) in E17.5 lungs from Prx1 WT and Prx1 null mice. E , Real-time PCR for plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in E17.5 lungs from Prx1 WT and Prx1 null mice. For Western immunoblotting ( B , D ), β-actin was used as a loading control. For mRNA expression by real-time PCR ( C , E ), target gene expression was normalized to GAPDH. Two asterisks indicate P
    Figure Legend Snippet: Prx1 regulates transforming growth factor (TGF)–β signaling in embryonic lungs. A , Immunohistochemical staining for TGF-β1 and TGF-β2 in lung sections derived from E15.5 ( top ) and D0 ( bottom ) Prx1 WT ( left ) and Prx1 null ( right ) mice. Brown indicates positive staining of antibodies. While TGF-β localization is restricted to the developing vessel area (arrows) in Prx1 WT lungs, in Prx1 null lungs TGF-β expression is observed in areas other than the vessel wall vicinity (arrowheads). Hematoxylin staining (blue) is used for nuclear counterstaining. Scale bar = 40 μm. B , Western blotting for TGF-β1 protein expression in E17.5 lungs from Prx1 WT and Prx1 null mice. C , Real-time polymerase chain reaction (PCR) for TGF-β1 messenger RNA (mRNA) expression in E17.5 lungs from Prx1 WT and Prx1 null mice. D , Western blotting for phosphorylated Smad2 (P-Smad2; top ) and total Smad 2/3 ( middle ) in E17.5 lungs from Prx1 WT and Prx1 null mice. E , Real-time PCR for plasminogen activator inhibitor type 1 (PAI-1) mRNA expression in E17.5 lungs from Prx1 WT and Prx1 null mice. For Western immunoblotting ( B , D ), β-actin was used as a loading control. For mRNA expression by real-time PCR ( C , E ), target gene expression was normalized to GAPDH. Two asterisks indicate P

    Techniques Used: Immunohistochemistry, Staining, Derivative Assay, Mouse Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Prx1 controls expression of tropoelastin (TE), fibrillin (Fbn) 1, and Fbn2 during lung vascular development. A , Immunohistochemical staining for TE, Fbn1, and Fbn2 in lung sections derived from E15.5 Prx1 WT ( left ) and Prx1 null ( right ) mice. Arrows indicate developing vessels, in which TE expression is observed in Prx1 WT mice but not in Prx1 null mice. Brown color indicates positive staining; hematoxylin staining in blue is nuclear counterstaining. B , Western immunoblotting for Fbn1, Fbn2, and TE protein in Prx1 WT and Prx1 null lungs isolated at E17.5. β-Actin was used as a loading control. C , Real-time polymerase chain reaction for TE and Fbn2 messenger RNA expression in Prx1 WT and Prx1 null lungs isolated at E17.5. Target gene expression was normalized to GAPDH expression. One asterisk indicates P
    Figure Legend Snippet: Prx1 controls expression of tropoelastin (TE), fibrillin (Fbn) 1, and Fbn2 during lung vascular development. A , Immunohistochemical staining for TE, Fbn1, and Fbn2 in lung sections derived from E15.5 Prx1 WT ( left ) and Prx1 null ( right ) mice. Arrows indicate developing vessels, in which TE expression is observed in Prx1 WT mice but not in Prx1 null mice. Brown color indicates positive staining; hematoxylin staining in blue is nuclear counterstaining. B , Western immunoblotting for Fbn1, Fbn2, and TE protein in Prx1 WT and Prx1 null lungs isolated at E17.5. β-Actin was used as a loading control. C , Real-time polymerase chain reaction for TE and Fbn2 messenger RNA expression in Prx1 WT and Prx1 null lungs isolated at E17.5. Target gene expression was normalized to GAPDH expression. One asterisk indicates P

    Techniques Used: Expressing, Immunohistochemistry, Staining, Derivative Assay, Mouse Assay, Western Blot, Isolation, Real-time Polymerase Chain Reaction, RNA Expression

    Prx1 controls expression of elastic extracellular matrix (ECM) components. A , Quantification of elastic ECM protein expression by densitometry Western blotting analysis in ImageJ. All target protein values were standardized by β-actin intensity. B – D , Real-time polymerase chain reaction for Prx1 ( B ), tropoelastin (ELN; C ), and fibrillin (Fbn) 1 ( D ) messenger RNA expression in 10T1/2 cells transiently transfected with empty or Prx1-containing construct. Target gene expression was normalized to cyclophilin A (Cyclo A) expression. One asterisk indicates P
    Figure Legend Snippet: Prx1 controls expression of elastic extracellular matrix (ECM) components. A , Quantification of elastic ECM protein expression by densitometry Western blotting analysis in ImageJ. All target protein values were standardized by β-actin intensity. B – D , Real-time polymerase chain reaction for Prx1 ( B ), tropoelastin (ELN; C ), and fibrillin (Fbn) 1 ( D ) messenger RNA expression in 10T1/2 cells transiently transfected with empty or Prx1-containing construct. Target gene expression was normalized to cyclophilin A (Cyclo A) expression. One asterisk indicates P

    Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, RNA Expression, Transfection, Construct

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    Western Blot:

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    Article Snippet: .. Western blots were probed for cyclophilin B with rabbit polyclonal antibody (Affinity BioReagents) or mouse monoclonal antibody (k2E2; Abcam), type I collagen (SouthernBioTech), P3H1 (Abnova), PDI (Stressgen), Hsp47 (Stressgen), FKBP65 (BD), GST (Sigma), CRTAP (Abnova) and beta-actin (Sigma). .. Reactive bands were visualized with a secondary antibody conjugated with HRP (Zymed) and chemiluminescence (Thermo scientific).

    Incubation:

    Article Title: Control of Hepatitis C Virus Replication in Mouse Liver-Derived Cells by MAVS-Dependent Production of Type I and Type III Interferons
    Article Snippet: .. The membrane was then incubated with either anti-NS3337 tag (1:300), anti-HA tag (1:1,000; Covance), anti-FLAG tag (1:1,000; Sigma-Aldrich), anti-hMAVS or anti-mMAVS (both at 1:300; Santa Cruz), anti-green fluorescent protein (anti-GFP, 1:1,000; Santa Cruz), or anti-β-actin (1:1,000; Sigma-Aldrich), followed by incubation with secondary antibody coupled to horseradish peroxidase (Sigma-Aldrich). .. Bound antibodies were detected with the ECL Plus detection system (GE Healthcare).

    other:

    Article Title: Enterovirus 3A Facilitates Viral Replication by Promoting Phosphatidylinositol 4-Kinase IIIβ–ACBD3 Interaction
    Article Snippet: Antibodies against Flag, GFP, Myc, and β-actin were purchased from Sigma-Aldrich (St. Louis, MO).

    Article Title: ApoE Receptor 2 Regulates Synapse and Dendritic Spine Formation
    Article Snippet: Antibodies We used antibodies anti-HA (Abcam), anti-X11α (BD Bioscience, Sigma), anti-Flag (Sigma), anti-PSD-95 (Chemicon), anti-GFP (Invitrogen), β-actin (Chemicon), anti-ApoEr2 (Sigma), anti-GluA1 (Chemicon), anti-GluA2 (Chemicon), anti-c-myc (Abcam), and anti-synaptophysin (Sigma).

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    Millipore microglial sa β gal activity
    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase <t>(SA-β-gal)</t> activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p
    Microglial Sa β Gal Activity, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore β actin
    Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or <t>anti-β-actin</t> primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.
    β Actin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2095 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: Microglia change from a reactive to an age-like phenotype with the time in culture

    doi: 10.3389/fncel.2014.00152

    Figure Lengend Snippet: Microglia aged in culture display signs of senescence, including increased senescent-associated β-galactosidase (SA-β-gal) activity and microRNA (miR)-146a expression. Microglial cells were kept in culture for 2 and 16 days in vitro (DIV). Activity of SA-β-gal was determined using a commercial kit. (A) Representative images of 2 and 16 DIV microglia showing SA-β-gal staining. (B) SA-β-gal-positive cells were counted and results expressed in graph bars as mean ± SEM. (C) miR-146a expression was evaluated by Real-Time PCR. Results are expressed in graph bars as mean ± SEM. Cultures, n = 4 per group. t -test, * p

    Article Snippet: Microglial SA-β-gal activity was determined using the Cellular senescence assay kit (Millipore), according to the manufacturer instructions.

    Techniques: Activity Assay, Expressing, In Vitro, Staining, Real-time Polymerase Chain Reaction

    Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or anti-β-actin primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.

    Journal: PLoS ONE

    Article Title: Physiological ER Stress Mediates the Differentiation of Fibroblasts

    doi: 10.1371/journal.pone.0123578

    Figure Lengend Snippet: Effects of repeated TM stimulation on fibroblasts’ morphology and GRP78/BiP expression. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. After this repeated TM or DMSO stimulation, medium was changed to DMEM with 2% horse serum (Basal medium condition) and incubated for 12h to induce differentiation. Primary cultured fibroblasts were treated with 1μg/ml TM or DMSO for 5 minutes per day 3 days in series. (a) Just after this repeated TM or DMSO stimulation, the cells were observed (upper panels) and stained by anti-Bip antibody (bottom panels). (b) The cells treated with TM (Rep-TM) or DMSO (Rep-DM/Rep-DMSO) cultured in the culture condition medium (C.C.) or in the Basal medium condition for differentiation (M.C.) were collected and lysed. Western blot analysis was performed using an anti-Bip or anti-β-actin primary antibody (upper panels). Quantitative data were obtained by densitometry of the bands. Data are expressed as the mean ± SEM for at least three independent experiments (shown as a ratio of the Rep-DM C.C.). The P value was compared with the control and calculated by Student's T test. (c) Left and middle panels show the cells treated with TM (Rep-TM) or DMSO (Rep-DMSO) cultured at Basal medium condition. Right panel shows the cells treated with TGF-β1 after the incubation at the basal medium condition.

    Article Snippet: Equal amounts of protein were subjected to 5–20% gradient SDS-PAGE, e-PAGEL (ATTO CO., Tokyo, Japan) for GRP78/Bip or β-actin and transferred to PVDF membrane (Millipore, Bedford, MA).

    Techniques: Expressing, Cell Culture, Incubation, Staining, Western Blot

    Anti-proliferative effects of Dp44mT and the effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in OECM-1 cells. ( A ) OECM-1 cells were treated with various concentrations of Dp44mt as indicated for 24 h and growth inhibitory effect was determined by the CyQUANT cell proliferation assay. The data shown in each bar chart represent the mean percentage ± SE of cells in each dose of the iron chelators treatment and are compared with the control solvent-treated group ( n = 8). The OECM-1 cells were treated with various concentrations of Dp44mt ( B ), DFO ( C ), and deferasirox ( D ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group, + p

    Journal: International Journal of Molecular Sciences

    Article Title: The Iron Chelator, Dp44mT, Effectively Inhibits Human Oral Squamous Cell Carcinoma Cell Growth in Vitro and in Vivo

    doi: 10.3390/ijms17091435

    Figure Lengend Snippet: Anti-proliferative effects of Dp44mT and the effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in OECM-1 cells. ( A ) OECM-1 cells were treated with various concentrations of Dp44mt as indicated for 24 h and growth inhibitory effect was determined by the CyQUANT cell proliferation assay. The data shown in each bar chart represent the mean percentage ± SE of cells in each dose of the iron chelators treatment and are compared with the control solvent-treated group ( n = 8). The OECM-1 cells were treated with various concentrations of Dp44mt ( B ), DFO ( C ), and deferasirox ( D ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group, + p

    Article Snippet: The β-actin (MAB1501; Millipore, Temecula, CA, USA) was used as the internal positive control.

    Techniques: CyQUANT Assay, Proliferation Assay, Western Blot, Produced

    Effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in SAS cells. The SAS cells were treated with various concentrations of Dp44mt ( A ), DFO ( B ), and deferasirox ( C ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group ( D – F ), + p

    Journal: International Journal of Molecular Sciences

    Article Title: The Iron Chelator, Dp44mT, Effectively Inhibits Human Oral Squamous Cell Carcinoma Cell Growth in Vitro and in Vivo

    doi: 10.3390/ijms17091435

    Figure Lengend Snippet: Effects of Dp44mT, DFO, and deferasirox on protein expressions of NDRG1, NDRG2, NDRG3, Maspin, and cyclin D1 in SAS cells. The SAS cells were treated with various concentrations of Dp44mt ( A ), DFO ( B ), and deferasirox ( C ) as indicated for 24 h, the expressions of NDRG1, NDRG2, NDRG3, Maspin, cyclin D1 proteins, and β-actin were determined by Western-blot assays. Data of quantitative analysis were expressed as the intensity of protein bands produced from the expressions of the target genes/β-actin (±SE; n = 3) relative to the control solvent-treated group ( D – F ), + p

    Article Snippet: The β-actin (MAB1501; Millipore, Temecula, CA, USA) was used as the internal positive control.

    Techniques: Western Blot, Produced

    The Bmi1 siRNA delivery efficiency by FA-siRNA-L and repression effects on Bmi1 expression in KB cells. For western blotting and qRT-PCR assay, cells cultured in DMEM medium were harvested and protein or mRNA was extracted for the determinations. For the uptake study, KB and LO2 cells grown in a monolayer were incubated with FA-siRNA-L, siRNA-L, and Chol-siRNA for 1 h at 37°C and were then used for the next assays. A. The Bmi1 protein expression in cancer or normal cell lines detected by western blot. B. QRT-PCR quantitative analysis of the expression of Bmi1 in cancer cells in mRNA level. C. Cellular uptake and intracellular distribution of Bmi1 siRNA in the KB cells. D. Western blotting of Bmi1 expression in the cells treated with Bmi1 siRNA complex or liposomes. LO2 cells were used as the normal cell control and β-actin was used as the loading control. E. Quantitative analysis of the western blotting bands. Data are expressed as mean ± SD of 3 independent samples. **: P

    Journal: Theranostics

    Article Title: Co-delivery of Doxorubicin and Bmi1 siRNA by Folate Receptor Targeted Liposomes Exhibits Enhanced Anti-Tumor Effects in vitro and in vivo

    doi: 10.7150/thno.9423

    Figure Lengend Snippet: The Bmi1 siRNA delivery efficiency by FA-siRNA-L and repression effects on Bmi1 expression in KB cells. For western blotting and qRT-PCR assay, cells cultured in DMEM medium were harvested and protein or mRNA was extracted for the determinations. For the uptake study, KB and LO2 cells grown in a monolayer were incubated with FA-siRNA-L, siRNA-L, and Chol-siRNA for 1 h at 37°C and were then used for the next assays. A. The Bmi1 protein expression in cancer or normal cell lines detected by western blot. B. QRT-PCR quantitative analysis of the expression of Bmi1 in cancer cells in mRNA level. C. Cellular uptake and intracellular distribution of Bmi1 siRNA in the KB cells. D. Western blotting of Bmi1 expression in the cells treated with Bmi1 siRNA complex or liposomes. LO2 cells were used as the normal cell control and β-actin was used as the loading control. E. Quantitative analysis of the western blotting bands. Data are expressed as mean ± SD of 3 independent samples. **: P

    Article Snippet: Membranes were blocked in 5% skim milk for 1 h and then incubated with monoclonal antibody against Bmi1 (1:1000, Millipore, USA) or β-actin (1:5000, Millipore, USA) overnight.

    Techniques: Expressing, Western Blot, Quantitative RT-PCR, Cell Culture, Incubation