anti polyglutamine expansion diseases marker antibody  (Millipore)


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    Structured Review

    Millipore anti polyglutamine expansion diseases marker antibody
    Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient <t>(polyQ</t> expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb
    Anti Polyglutamine Expansion Diseases Marker Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 701 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration"

    Article Title: Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06041-3

    Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient (polyQ expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb
    Figure Legend Snippet: Staufen1 protein but not mRNA steady-state levels are increased in neurodegenerative disease cells and tissues. Western blot analysis of SCA2- FBs ( a ) and LBCs ( b ) show increased STAU1 levels compared with normal controls. DDX6 levels are unchanged. HD and SCA3 patient (polyQ expanded) FBs were used as additional controls. Four normal and five SCA2 FBs, and two normal and three SCA2 LBCs were used. c , d Western blot analyses of ATXN2 Q127 ( c ) and BAC-Q72 ( d ) mouse cerebellar extracts (24 weeks of age) showing increased Stau1 levels compared with wild-type or BAC-Q22 controls ( n = 2–3 animals per group). e Western blot of FB extracts from an ALS patient with the TDP-43 G298S mutation show increased STAU1 levels. β-Actin was used as loading control and representative blots of three independent experiments are shown. f – h STAU1 RNA levels are unaltered in SCA2 and ALS cells and SCA2 mice. qRT-PCR analyses of STAU1 mRNA in SCA2 FBs and ALS FB with TDP-43 G298S mutation ( f ) or SCA2 LBCs ( g ). h qRT-PCR analyses of cerebellar RNAs from ATXN2 Q127 and BAC-Q72 mice compared to wild-type littermates (24 weeks of age; n = animals per group). Gene expression levels were normalized to Actb

    Techniques Used: Western Blot, BAC Assay, Mutagenesis, Mouse Assay, Quantitative RT-PCR, Expressing

    2) Product Images from "Reduced synaptic function of Kainate receptors in the insular cortex of Fmr1 Knock-out mice"

    Article Title: Reduced synaptic function of Kainate receptors in the insular cortex of Fmr1 Knock-out mice

    Journal: Molecular Brain

    doi: 10.1186/s13041-018-0396-1

    The expression of KARs in the cultured cortical neurons from Fmr1 WT and Fmr1 KO mice. a , b , The abundance of GluK1 or GluK2/3 in the homogenate of the cultured cortical neurons from Fmr1 WT mice and Fmr1 KO mice showed no change. c , d , Surface expression levels of GluK2/3 in insular cortex neurons obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot analysis. The surface expression levels of GluK2/3 were not altered between Fmr1 WT and Fmr1 KO mice (n = 3 independent experiments). Actin was used as negative control for surface biotinylation
    Figure Legend Snippet: The expression of KARs in the cultured cortical neurons from Fmr1 WT and Fmr1 KO mice. a , b , The abundance of GluK1 or GluK2/3 in the homogenate of the cultured cortical neurons from Fmr1 WT mice and Fmr1 KO mice showed no change. c , d , Surface expression levels of GluK2/3 in insular cortex neurons obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot analysis. The surface expression levels of GluK2/3 were not altered between Fmr1 WT and Fmr1 KO mice (n = 3 independent experiments). Actin was used as negative control for surface biotinylation

    Techniques Used: Expressing, Cell Culture, Mouse Assay, Western Blot, Negative Control

    Distribution of KARs in the insular cortex from Fmr1 WT mice. Presynaptic marker synaptophysin, postsynaptic marker PSD95, NMDAR subunits GluN2B and GluN2A, KAR subunits GluK1 and GluK2/3 or synaptic protein Rab5 were analyzed by Western blot in the homogenates (H1, 10 μg), postnuclear supernatant (S1, 5 μg), nuclei and large debris pellet (P1, 10 μg), cytosomes (S2, 5 μg), crude synaptosomal membrane (P2, 5 μg), non-PSD (5 μg) or PSD (10 μg) fractions of the insular cortex in Fmr1 WT mice. This experiment was repeated three times
    Figure Legend Snippet: Distribution of KARs in the insular cortex from Fmr1 WT mice. Presynaptic marker synaptophysin, postsynaptic marker PSD95, NMDAR subunits GluN2B and GluN2A, KAR subunits GluK1 and GluK2/3 or synaptic protein Rab5 were analyzed by Western blot in the homogenates (H1, 10 μg), postnuclear supernatant (S1, 5 μg), nuclei and large debris pellet (P1, 10 μg), cytosomes (S2, 5 μg), crude synaptosomal membrane (P2, 5 μg), non-PSD (5 μg) or PSD (10 μg) fractions of the insular cortex in Fmr1 WT mice. This experiment was repeated three times

    Techniques Used: Mouse Assay, Marker, Western Blot

    The abundance of KARs in the synaptosome is decreased in Fmr1 KO mice. a , b , Total expression levels of GluK1 and GluK2/3 in the homogenates fraction of the insular cortex conducted from Fmr1 WT and Fmr1 KO mice were detected by Western blot. The expression levels of GluK1 and GluK2/3 in the homogenates were not altered between Fmr1 WT and Fmr1 KO mice ( n = 3 mice for each group). c , d , Expression levels of GluK1 and GluK2/3 in the synaptosome of the insular cortex obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot anaylsis. The expression levels of GluK1 and GluK2/3 in the homogenates was significantly reduced in Fmr1 KO mice compare to Fmr1 WT mice ( n = 5 mice for each group). * P
    Figure Legend Snippet: The abundance of KARs in the synaptosome is decreased in Fmr1 KO mice. a , b , Total expression levels of GluK1 and GluK2/3 in the homogenates fraction of the insular cortex conducted from Fmr1 WT and Fmr1 KO mice were detected by Western blot. The expression levels of GluK1 and GluK2/3 in the homogenates were not altered between Fmr1 WT and Fmr1 KO mice ( n = 3 mice for each group). c , d , Expression levels of GluK1 and GluK2/3 in the synaptosome of the insular cortex obtained from Fmr1 WT and Fmr1 KO mice were detected by western blot anaylsis. The expression levels of GluK1 and GluK2/3 in the homogenates was significantly reduced in Fmr1 KO mice compare to Fmr1 WT mice ( n = 5 mice for each group). * P

    Techniques Used: Mouse Assay, Expressing, Western Blot

    3) Product Images from "Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss)"

    Article Title: Piscine orthoreovirus subtype 3 (PRV-3) causes heart inflammation in rainbow trout (Oncorhynchus mykiss)

    Journal: Veterinary Research

    doi: 10.1186/s13567-019-0632-4

    Purified PRV-3 particles. A Transmission electron microscopy (TEM) of purified PRV-3 viral particles. B Western blot analysis of proteins from purified PRV-3 particles using antiserum raised against PRV-1 σ1, σ3 and µ1 proteins. Observed bands correspond to predicted full length proteins of 35 kDa, 37 kDA and 74 kDa, respectively.
    Figure Legend Snippet: Purified PRV-3 particles. A Transmission electron microscopy (TEM) of purified PRV-3 viral particles. B Western blot analysis of proteins from purified PRV-3 particles using antiserum raised against PRV-1 σ1, σ3 and µ1 proteins. Observed bands correspond to predicted full length proteins of 35 kDa, 37 kDA and 74 kDa, respectively.

    Techniques Used: Purification, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Western Blot

    4) Product Images from "Mural cell-derived laminin-α5 plays a detrimental role in ischemic stroke"

    Article Title: Mural cell-derived laminin-α5 plays a detrimental role in ischemic stroke

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-019-0676-8

    Lama5 expression is abrogated in mural cells in α5-PKO mice. a Representative images of laminin-α2 (green) and laminin-α5 (red) staining in the cortex of control and α5-PKO mice. Scale bar = 100 μm. b Representative western blotting and quantification of laminin-α2 and laminin-α5 levels in the cortex of control and α5-PKO mice. n = 4. c Representative images of smooth muscle actin-α (SMA, green) and laminin-α5 (red) staining in primary mural cells isolated from control and α5-PKO brains. Scale bar = 50 μm. d Quantification showing the lack of laminin-α5 expression in primary mural cells isolated from α5-PKO brains. n = 6 independent experiments with at least 50 cells examined in each experiment. Student’s t -test, *** p
    Figure Legend Snippet: Lama5 expression is abrogated in mural cells in α5-PKO mice. a Representative images of laminin-α2 (green) and laminin-α5 (red) staining in the cortex of control and α5-PKO mice. Scale bar = 100 μm. b Representative western blotting and quantification of laminin-α2 and laminin-α5 levels in the cortex of control and α5-PKO mice. n = 4. c Representative images of smooth muscle actin-α (SMA, green) and laminin-α5 (red) staining in primary mural cells isolated from control and α5-PKO brains. Scale bar = 50 μm. d Quantification showing the lack of laminin-α5 expression in primary mural cells isolated from α5-PKO brains. n = 6 independent experiments with at least 50 cells examined in each experiment. Student’s t -test, *** p

    Techniques Used: Expressing, Mouse Assay, Staining, Western Blot, Isolation

    5) Product Images from "Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction"

    Article Title: Excess Podocyte Semaphorin-3A Leads to Glomerular Disease Involving PlexinA1–Nephrin Interaction

    Journal: The American Journal of Pathology

    doi: 10.1016/j.ajpath.2013.06.022

    Sema3a gain-of-function down-regulates nephrin expression. A : Quantitative PCR indicates decreased nephrin mRNA and stable podocin mRNA in Sema3a -overexpressing mice. B and C : Western blotting for nephrin ( B ) and podocin ( C ) in whole-kidney lysate indicates that excess Sema3a induces reversible nephrin down-regulation but does not affect podocin expression. Quantitation is based on at least four independent experiments. D : Immunofluorescence reveals down-regulation of nephrin and stable expression of podocin localized to glomerular podocytes. E : Immunofluorescence reveals no change in podocyte number (WT1 + nuclei) in Sema3a -overexpressing mice versus controls. DAPI labels nuclei; merged images confirm nuclear WT1 staining. F : Quantitation of WT1 + cells per standard area. Data are expressed as means ± SEM. n = 3 mice per experimental group (approximately 20 glomeruli quantified per mouse). Scale bars: 100 μm ( D ); 20 μm ( E ).
    Figure Legend Snippet: Sema3a gain-of-function down-regulates nephrin expression. A : Quantitative PCR indicates decreased nephrin mRNA and stable podocin mRNA in Sema3a -overexpressing mice. B and C : Western blotting for nephrin ( B ) and podocin ( C ) in whole-kidney lysate indicates that excess Sema3a induces reversible nephrin down-regulation but does not affect podocin expression. Quantitation is based on at least four independent experiments. D : Immunofluorescence reveals down-regulation of nephrin and stable expression of podocin localized to glomerular podocytes. E : Immunofluorescence reveals no change in podocyte number (WT1 + nuclei) in Sema3a -overexpressing mice versus controls. DAPI labels nuclei; merged images confirm nuclear WT1 staining. F : Quantitation of WT1 + cells per standard area. Data are expressed as means ± SEM. n = 3 mice per experimental group (approximately 20 glomeruli quantified per mouse). Scale bars: 100 μm ( D ); 20 μm ( E ).

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

    Podocyte Sema3a gain-of-function induces mesangial expansion foot process effacement and endothelial injury. A and B : PAS staining reveals normal glomeruli in kidneys of single-transgenic mice with doxycycline (+dox) and in uninduced Sema3a + mice (−dox). C and D : Two representative Sema3a-overexpressing glomeruli (+dox) exhibit mesangial expansion. E – H : TEM reveals normal glomerular filtration barrier ultrastructure in uninduced Sema3a + kidneys ( E and G ). Sema3a overexpression induces foot process effacement, endothelial cell swelling, and marked GBM expansion ( F and H ). Original magnification, ×400 ( A–D ). Scale bars: 1 μm ( E and F ) and 500 nm ( G and H ).
    Figure Legend Snippet: Podocyte Sema3a gain-of-function induces mesangial expansion foot process effacement and endothelial injury. A and B : PAS staining reveals normal glomeruli in kidneys of single-transgenic mice with doxycycline (+dox) and in uninduced Sema3a + mice (−dox). C and D : Two representative Sema3a-overexpressing glomeruli (+dox) exhibit mesangial expansion. E – H : TEM reveals normal glomerular filtration barrier ultrastructure in uninduced Sema3a + kidneys ( E and G ). Sema3a overexpression induces foot process effacement, endothelial cell swelling, and marked GBM expansion ( F and H ). Original magnification, ×400 ( A–D ). Scale bars: 1 μm ( E and F ) and 500 nm ( G and H ).

    Techniques Used: Staining, Transgenic Assay, Mouse Assay, Transmission Electron Microscopy, Filtration, Over Expression

    Sema3a decreases αvβ3 integrin glomerular activity and induces podocyte contraction and shape change. A : Western blotting and quantitation indicate that αvβ3, β3, and β1 integrin whole-kidney expression levels were not changed by Sema3a overexpression. B : Immunofluorescence reveals decrease of both glomerular total αvβ3 integrin and Wow-1 (active αvβ3 integrin) in Sema3a -overexpressing kidneys. The αvβ3 integrin and nephrin partially colocalize; negative controls demonstrate no signal in the absence of the primary antibodies (total αvβ3 integrin, nephrin, and active αvβ3 integrin). C ) indicates an approximately 50% decrease in αvβ3 integrin activity. D : Sema3a induced cell contraction and shape change in a dose-dependent manner; two representative images from each condition are shown (rhodamine phalloidin staining). Note decreasing F-actin staining at higher Sema3a concentration. E : Differentiated Sema3a podocytes exposed to vehicle or 50 ng/mL recombinant mouse Sema3a for 1 hour or 12 hours exhibited significant change in cell size. F : Quantitation after 12 hours of Sema3a exposure indicates dose-dependent decrease in cell area. G : Model for Sema3a-induced glomerular disease mechanism. Data are expressed as means ± SEM, representative of four independent experiments (approximately 50 cells per experiment). ∗ P
    Figure Legend Snippet: Sema3a decreases αvβ3 integrin glomerular activity and induces podocyte contraction and shape change. A : Western blotting and quantitation indicate that αvβ3, β3, and β1 integrin whole-kidney expression levels were not changed by Sema3a overexpression. B : Immunofluorescence reveals decrease of both glomerular total αvβ3 integrin and Wow-1 (active αvβ3 integrin) in Sema3a -overexpressing kidneys. The αvβ3 integrin and nephrin partially colocalize; negative controls demonstrate no signal in the absence of the primary antibodies (total αvβ3 integrin, nephrin, and active αvβ3 integrin). C ) indicates an approximately 50% decrease in αvβ3 integrin activity. D : Sema3a induced cell contraction and shape change in a dose-dependent manner; two representative images from each condition are shown (rhodamine phalloidin staining). Note decreasing F-actin staining at higher Sema3a concentration. E : Differentiated Sema3a podocytes exposed to vehicle or 50 ng/mL recombinant mouse Sema3a for 1 hour or 12 hours exhibited significant change in cell size. F : Quantitation after 12 hours of Sema3a exposure indicates dose-dependent decrease in cell area. G : Model for Sema3a-induced glomerular disease mechanism. Data are expressed as means ± SEM, representative of four independent experiments (approximately 50 cells per experiment). ∗ P

    Techniques Used: Activity Assay, Western Blot, Quantitation Assay, Expressing, Over Expression, Immunofluorescence, Staining, Concentration Assay, Recombinant

    Sema3a-induced glomerular ultrastructural abnormalities are reversible. A and D : TEM reveals normal glomerular filtration barrier ultrastructure in uninduced Sema3a + kidneys. B and E : Sema3a podocyte overexpression induces foot process effacement, mesangial matrix expansion, and GBM lamination. C and F : At 2 weeks after doxycycline removal (+/−dox), the Sema3a-induced foot process effacement, GBM, and endothelial cell abnormalities resolve. G : Morphometry confirms reversibility of foot process effacement. Data are expressed as means ± SEM. ∗ P
    Figure Legend Snippet: Sema3a-induced glomerular ultrastructural abnormalities are reversible. A and D : TEM reveals normal glomerular filtration barrier ultrastructure in uninduced Sema3a + kidneys. B and E : Sema3a podocyte overexpression induces foot process effacement, mesangial matrix expansion, and GBM lamination. C and F : At 2 weeks after doxycycline removal (+/−dox), the Sema3a-induced foot process effacement, GBM, and endothelial cell abnormalities resolve. G : Morphometry confirms reversibility of foot process effacement. Data are expressed as means ± SEM. ∗ P

    Techniques Used: Transmission Electron Microscopy, Filtration, Over Expression

    Podocyte Sema3a gain-of-function induces proteinuria. A : Western blotting indicates that Sema3a + mice overexpress kidney Sema3a approximately twofold after doxycycline-induction for 1 month. Smaller molecular weight bands represent cleaved Sema3a. Quantitation is based on at least three independent experiments. B : Dual immunofluorescence reveals Sema3a (red) and podocin (green) expression in developing mouse glomeruli; colocalization (yellow) indicates Sema3a expression in podocytes. C and D : Western blotting ( C ) and Coomassie Blue gel ( D ) indicate that excess Sema3a induces reversible proteinuria, based on albumin detected in urine samples corrected for creatinine. E and F : Excess Sema3a does not alter creatinine clearance ( E ) or blood pressure ( F ) within 1 month. Data are expressed as means ± SEM. White bars, control (−dox); black bars, Sema3a excess (+dox); gray bars, reversibility (+/−dox). ∗ P
    Figure Legend Snippet: Podocyte Sema3a gain-of-function induces proteinuria. A : Western blotting indicates that Sema3a + mice overexpress kidney Sema3a approximately twofold after doxycycline-induction for 1 month. Smaller molecular weight bands represent cleaved Sema3a. Quantitation is based on at least three independent experiments. B : Dual immunofluorescence reveals Sema3a (red) and podocin (green) expression in developing mouse glomeruli; colocalization (yellow) indicates Sema3a expression in podocytes. C and D : Western blotting ( C ) and Coomassie Blue gel ( D ) indicate that excess Sema3a induces reversible proteinuria, based on albumin detected in urine samples corrected for creatinine. E and F : Excess Sema3a does not alter creatinine clearance ( E ) or blood pressure ( F ) within 1 month. Data are expressed as means ± SEM. White bars, control (−dox); black bars, Sema3a excess (+dox); gray bars, reversibility (+/−dox). ∗ P

    Techniques Used: Western Blot, Mouse Assay, Molecular Weight, Quantitation Assay, Immunofluorescence, Expressing

    Sema3a signaling receptor plexinA 1 interacts with nephrin. A : Representative Western blots and quantitation indicate no significant changes in plexinA 1 or neuropilin 1 expression in whole kidneys after doxycycline induction. B : Immunofluorescence reveals colocalization of plexinA 1 and nephrin in cultured podocytes. C : Co-immunoprecipitation reveals association of endogenous plexinA 1 and FLAG-nephrin. Lane 1, cultured podocytes; lane 2, whole kidney. D : Reciprocal nephrin and plexinA 1 coimmunoprecipitation reveals nephrin–plexinA 1 interaction in transfected HEK cells. E and F : GST binding assay ( E ) indicates direct interaction of purified FLAG-plexinA 1 with nephrin cytoplasmic domain (GST-CD-nephrin, approximately 60 kDa; red arrowhead ); the GST-control is approximately 25 kDa ( black arrowhead ). Overlay assay ( F ) indicates that plexinA 1 –nephrin interaction is direct. Purified FLAG-plexinA 1 binds increasing amounts of GST-CD-nephrin blotted on cellulose membrane, as detected by FLAG immunoblotting; the GST Western blot confirms equal loading. Data are representative of at least three independent experiments. IP, immunoprecipitation; RS, rabbit serum; WB, Western blot; WCL, whole-cell lysate. Original magnification, ×400 ( B ).
    Figure Legend Snippet: Sema3a signaling receptor plexinA 1 interacts with nephrin. A : Representative Western blots and quantitation indicate no significant changes in plexinA 1 or neuropilin 1 expression in whole kidneys after doxycycline induction. B : Immunofluorescence reveals colocalization of plexinA 1 and nephrin in cultured podocytes. C : Co-immunoprecipitation reveals association of endogenous plexinA 1 and FLAG-nephrin. Lane 1, cultured podocytes; lane 2, whole kidney. D : Reciprocal nephrin and plexinA 1 coimmunoprecipitation reveals nephrin–plexinA 1 interaction in transfected HEK cells. E and F : GST binding assay ( E ) indicates direct interaction of purified FLAG-plexinA 1 with nephrin cytoplasmic domain (GST-CD-nephrin, approximately 60 kDa; red arrowhead ); the GST-control is approximately 25 kDa ( black arrowhead ). Overlay assay ( F ) indicates that plexinA 1 –nephrin interaction is direct. Purified FLAG-plexinA 1 binds increasing amounts of GST-CD-nephrin blotted on cellulose membrane, as detected by FLAG immunoblotting; the GST Western blot confirms equal loading. Data are representative of at least three independent experiments. IP, immunoprecipitation; RS, rabbit serum; WB, Western blot; WCL, whole-cell lysate. Original magnification, ×400 ( B ).

    Techniques Used: Western Blot, Quantitation Assay, Expressing, Immunofluorescence, Cell Culture, Immunoprecipitation, Transfection, Binding Assay, Purification, Overlay Assay

    6) Product Images from "MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice"

    Article Title: MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003280

    Western blot analysis of MMR and DHFR protein levels. MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods . B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.
    Figure Legend Snippet: Western blot analysis of MMR and DHFR protein levels. MMR expression in liver and striatum from 4 and 16 week-old mouse. Actin was used as a loading control. MSH2: 104 kD, MSH6: 160 kD, MSH3: 127 kD (Ab = 2F11) and actin: 42 kD. DHFR expression in cortex from 4 and 16 week-old mice DHFR: 21 kD. A) Simultaneous Western blot using MSH2-, MSH3-, MSH6- and actin-specific antibodies in liver. For antibody dilutions see Materials and methods . B) Western blot using only anti-MSH3 (Ab = 2F11) and actin antibodies in liver and striatum. C) Western blot analysis of DHFR in cortex from 4 and 16 week-old mice.

    Techniques Used: Western Blot, Expressing, Mouse Assay

    7) Product Images from "Antagonism of Bradykinin B2 Receptor Prevents Inflammatory Responses in Human Endothelial Cells by Quenching the NF-kB Pathway Activation"

    Article Title: Antagonism of Bradykinin B2 Receptor Prevents Inflammatory Responses in Human Endothelial Cells by Quenching the NF-kB Pathway Activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0084358

    BK stimulates translocation/phosphorylation of NF-κB in circulating proangiogenic cells. (A) p65 (NF-κB) phosphorylation following exposure to BK (1 µM, 15 min) in presence/absence of fasitibant (0.1 µM). Gel are representative of three experiments. The ratio between p-p65 over p65 is reported. *p
    Figure Legend Snippet: BK stimulates translocation/phosphorylation of NF-κB in circulating proangiogenic cells. (A) p65 (NF-κB) phosphorylation following exposure to BK (1 µM, 15 min) in presence/absence of fasitibant (0.1 µM). Gel are representative of three experiments. The ratio between p-p65 over p65 is reported. *p

    Techniques Used: Translocation Assay

    8) Product Images from "Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells"

    Article Title: Cinnamic acid induces apoptotic cell death and cytoskeleton disruption in human melanoma cells

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/1756-9966-32-31

    Cytoskeleton organization in NGM cells treated with 3.2 mM cinnamic acid. The cells were treated with the drug for 48 hours. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). The images were obtained by Laser Scanning Confocal Microscopy. Note that there are cells with normal cytoskeletal organization (left column) and cells with drastic morphological changes (intermediate and right columns).
    Figure Legend Snippet: Cytoskeleton organization in NGM cells treated with 3.2 mM cinnamic acid. The cells were treated with the drug for 48 hours. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). The images were obtained by Laser Scanning Confocal Microscopy. Note that there are cells with normal cytoskeletal organization (left column) and cells with drastic morphological changes (intermediate and right columns).

    Techniques Used: Staining, Labeling, Confocal Microscopy

    M30 and tubulin labeling in HT-144 cells. HT-144 cells were treated with 0.4 or 3.2 mM cinnamic acid for 24 or 48 hours. Fragmented cytokeratin 18 (green) were labeled with M30 antibody FITC and microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody TRITC-conjugated. A,B) cells with intact microtubules and M30(+); C) cells with microtubule disruption and M30(+); D) cells with microtubule disruption and M30(–). Arrows = M30 staining. The results demonstrate that cell death and microtubule disorganization are independent events in our system. The images were obtained by Laser Scanning Confocal Microscopy.
    Figure Legend Snippet: M30 and tubulin labeling in HT-144 cells. HT-144 cells were treated with 0.4 or 3.2 mM cinnamic acid for 24 or 48 hours. Fragmented cytokeratin 18 (green) were labeled with M30 antibody FITC and microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody TRITC-conjugated. A,B) cells with intact microtubules and M30(+); C) cells with microtubule disruption and M30(+); D) cells with microtubule disruption and M30(–). Arrows = M30 staining. The results demonstrate that cell death and microtubule disorganization are independent events in our system. The images were obtained by Laser Scanning Confocal Microscopy.

    Techniques Used: Labeling, Staining, Confocal Microscopy

    Cytoskeleton organization in NGM control cells. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). Note the stress fiber formation (actin filaments). The cells showed a microtubule network that was very finely departed from the centrosome region near the nucleus. We can also observe a mitotic cell (right column). The images were obtained by Laser Scanning Confocal Microscopy.
    Figure Legend Snippet: Cytoskeleton organization in NGM control cells. F-actin (green) was stained with phalloidin FITC-conjugated. Microtubules (blue) were labeled with anti-α and β tubulin and secondary antibody CY-5-conjugated. DNA was counterstained with propidium iodide (red). Note the stress fiber formation (actin filaments). The cells showed a microtubule network that was very finely departed from the centrosome region near the nucleus. We can also observe a mitotic cell (right column). The images were obtained by Laser Scanning Confocal Microscopy.

    Techniques Used: Staining, Labeling, Confocal Microscopy

    9) Product Images from "RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis"

    Article Title: RNA Interference and Single Particle Tracking Analysis of Hepatitis C Virus Endocytosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000702

    HCV localizes to actin rich filopodia and stress fibers during entry. DiD-HCV was allowed to bind to Huh-7.5 cells on ice for 1 hour followed by temperature shift to 37°C. Cells were fixed at 30 minutes post temperature shift (A) or 60 minutes post temperature shift (B) and stained for actin using Alexa Fluor 488 phalloidin (A) or were expressing GFP-actin (B). Enlarged insets are shown on the right. Scale bars = 10 µm. A. Inset shows DiD-HCV association with filopodia (top) and cortical actin cytoskeleton. B. Inset shows DiD-HCV association with actin stress fibers along the plasma membrane.
    Figure Legend Snippet: HCV localizes to actin rich filopodia and stress fibers during entry. DiD-HCV was allowed to bind to Huh-7.5 cells on ice for 1 hour followed by temperature shift to 37°C. Cells were fixed at 30 minutes post temperature shift (A) or 60 minutes post temperature shift (B) and stained for actin using Alexa Fluor 488 phalloidin (A) or were expressing GFP-actin (B). Enlarged insets are shown on the right. Scale bars = 10 µm. A. Inset shows DiD-HCV association with filopodia (top) and cortical actin cytoskeleton. B. Inset shows DiD-HCV association with actin stress fibers along the plasma membrane.

    Techniques Used: Staining, Expressing

    10) Product Images from "Deficiency in the Multicopy Sycp3-Like X-Linked Genes Slx and Slxl1 Causes Major Defects in Spermatid Differentiation"

    Article Title: Deficiency in the Multicopy Sycp3-Like X-Linked Genes Slx and Slxl1 Causes Major Defects in Spermatid Differentiation

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E10-07-0601

    Transgenically delivered shSLX constructs lead to a dramatic decrease of SLX and SLXL1 proteins. (A) Western blot detection of SLX/SLXL1 proteins on WT (i.e., nontransgenic siblings), shSLX1 and shSLX2 adult testicular extracts, after separation by 12% PAGE. A double band (indicated by a star) can be observed when detecting with the anti-SLX/SLXL1 antibody. Actin antibody was used for normalization. (B) Left panel, Western-blot detection of SLX/SLXL1 proteins on WT, shSLX1, shSLX2, and shSLX1,2 adult testicular extracts, after separation by 4–12% gradient PAGE. The upper band (gray arrowhead) corresponds to SLX protein. The bottom band (black arrowhead) is SLXL1 protein. Similar results were obtained on purified round spermatid extracts from WT, shSLX1, and shSLX2 mice (right panel, same conditions). Actin antibody was used for normalization.
    Figure Legend Snippet: Transgenically delivered shSLX constructs lead to a dramatic decrease of SLX and SLXL1 proteins. (A) Western blot detection of SLX/SLXL1 proteins on WT (i.e., nontransgenic siblings), shSLX1 and shSLX2 adult testicular extracts, after separation by 12% PAGE. A double band (indicated by a star) can be observed when detecting with the anti-SLX/SLXL1 antibody. Actin antibody was used for normalization. (B) Left panel, Western-blot detection of SLX/SLXL1 proteins on WT, shSLX1, shSLX2, and shSLX1,2 adult testicular extracts, after separation by 4–12% gradient PAGE. The upper band (gray arrowhead) corresponds to SLX protein. The bottom band (black arrowhead) is SLXL1 protein. Similar results were obtained on purified round spermatid extracts from WT, shSLX1, and shSLX2 mice (right panel, same conditions). Actin antibody was used for normalization.

    Techniques Used: Construct, Western Blot, Polyacrylamide Gel Electrophoresis, Purification, Mouse Assay

    Transcriptome analysis of Slx/Slxl1 -deficient round spermatids versus WT round spermatids. (A) Bar graph representing the number of genes found differentially expressed in round spermatids from Slx/Slxl1 -deficient males compared with those of WT males by microarray. Differentially expressed genes are shown by classification according to their likely biological function in Onto-Express. (B) Chromosome location of the genes found up- or down-regulated in case of Slx/Slxl1 deficiency. Note the enrichment of up-regulated genes on chromosome 7. This is due to the presence of the Klk1 genes that are affected by the loss of Slx/Slxl1 . (C) Quantification of the transcript level of the X-encoded Wdr13 gene in purified round spermatids of shSLY, SLX1, shSLX2, and WT males by real-time RT-PCR. Values were normalized to β- actin . One star indicates significant difference from WT value ( p
    Figure Legend Snippet: Transcriptome analysis of Slx/Slxl1 -deficient round spermatids versus WT round spermatids. (A) Bar graph representing the number of genes found differentially expressed in round spermatids from Slx/Slxl1 -deficient males compared with those of WT males by microarray. Differentially expressed genes are shown by classification according to their likely biological function in Onto-Express. (B) Chromosome location of the genes found up- or down-regulated in case of Slx/Slxl1 deficiency. Note the enrichment of up-regulated genes on chromosome 7. This is due to the presence of the Klk1 genes that are affected by the loss of Slx/Slxl1 . (C) Quantification of the transcript level of the X-encoded Wdr13 gene in purified round spermatids of shSLY, SLX1, shSLX2, and WT males by real-time RT-PCR. Values were normalized to β- actin . One star indicates significant difference from WT value ( p

    Techniques Used: Microarray, Purification, Quantitative RT-PCR

    Transgenically delivered shSLX constructs knockdown Slx and Slx-like1 at the transcript level. (A) Structure of the shSLX construct. The U6 promoter drives the expression of a short hairpin RNA targeting Slx/Slxl1 (shSLX). An insulator sequence is located upstream of the U6 promoter. A ‘genotyping’ tag (gTag) that is specific to each shSLX construct has been inserted to facilitate genotyping in double transgenic mice. (B) Quantification of the knockdown of Slx and Slxl1 transcripts in shSLX testes by real-time RT-PCR. Values were normalized to Acrv1 (an autosomal spermatid-specific gene). ‘ Slx-all ’ represents amplification with primers designed to amplify both Slx and Slxl1 transcripts. One star indicates significant difference from nontransgenic (WT) value ( p
    Figure Legend Snippet: Transgenically delivered shSLX constructs knockdown Slx and Slx-like1 at the transcript level. (A) Structure of the shSLX construct. The U6 promoter drives the expression of a short hairpin RNA targeting Slx/Slxl1 (shSLX). An insulator sequence is located upstream of the U6 promoter. A ‘genotyping’ tag (gTag) that is specific to each shSLX construct has been inserted to facilitate genotyping in double transgenic mice. (B) Quantification of the knockdown of Slx and Slxl1 transcripts in shSLX testes by real-time RT-PCR. Values were normalized to Acrv1 (an autosomal spermatid-specific gene). ‘ Slx-all ’ represents amplification with primers designed to amplify both Slx and Slxl1 transcripts. One star indicates significant difference from nontransgenic (WT) value ( p

    Techniques Used: Construct, Expressing, shRNA, Sequencing, Transgenic Assay, Mouse Assay, Quantitative RT-PCR, Amplification

    Impaired spermiogenesis and increased apoptosis of elongating spermatids in Slx/Slxl1 -deficient mice. (A–D) In PAS-stained stage XII testis tubules of Slx/Slxl1 -deficient (shSLX1 transgenic) mice, elongating spermatids are less developed than in controls (A). Typically, elongating spermatids with morphology of step 10 spermatids were observed in step XII tubules (C). In PAS-stained stage IX testis tubules of Slx/Slxl1 -deficient mice (D) mature sperm are retained in stage IX tubules, whereas in WT testes (B) mature sperm have already been released into the lumen. (E) PAS-stained stage X Slx/Slxl1 -deficient testis tubule. Several mature sperm are retained near the basal lamina (enlargement, right top corner). (F) PAS-stained stage IV Slx/Slxl1 -deficient testis tubule. A typical group of dying sperm organized in circle can be observed (enlargement, right top corner). (G–J) stage II-III testis tubules of Slx/Slxl1 -deficient mice present a great number of delayed elongating spermatids (marked with H4K12Ac antibody in green). Same stage tubules of control mice do not show any remaining H4K12Ac signal in spermatids. (K–M) Detection of apoptosis (TUNEL) in WT and Slx/Slxl1 -deficient testes. Representative pictures show the presence of several apoptotic elongating spermatids (in green) in a stage VI testis tubule of a Slx/Slxl1 -deficient male. No or very few apoptotic cells can be observed in a control testis (shSLX nontransgenic sibling). DAPI (in blue) was used to stain nuclei, and Lectin (in red) was used to stage the seminiferous tubules. Scale bar, 20 μm.
    Figure Legend Snippet: Impaired spermiogenesis and increased apoptosis of elongating spermatids in Slx/Slxl1 -deficient mice. (A–D) In PAS-stained stage XII testis tubules of Slx/Slxl1 -deficient (shSLX1 transgenic) mice, elongating spermatids are less developed than in controls (A). Typically, elongating spermatids with morphology of step 10 spermatids were observed in step XII tubules (C). In PAS-stained stage IX testis tubules of Slx/Slxl1 -deficient mice (D) mature sperm are retained in stage IX tubules, whereas in WT testes (B) mature sperm have already been released into the lumen. (E) PAS-stained stage X Slx/Slxl1 -deficient testis tubule. Several mature sperm are retained near the basal lamina (enlargement, right top corner). (F) PAS-stained stage IV Slx/Slxl1 -deficient testis tubule. A typical group of dying sperm organized in circle can be observed (enlargement, right top corner). (G–J) stage II-III testis tubules of Slx/Slxl1 -deficient mice present a great number of delayed elongating spermatids (marked with H4K12Ac antibody in green). Same stage tubules of control mice do not show any remaining H4K12Ac signal in spermatids. (K–M) Detection of apoptosis (TUNEL) in WT and Slx/Slxl1 -deficient testes. Representative pictures show the presence of several apoptotic elongating spermatids (in green) in a stage VI testis tubule of a Slx/Slxl1 -deficient male. No or very few apoptotic cells can be observed in a control testis (shSLX nontransgenic sibling). DAPI (in blue) was used to stain nuclei, and Lectin (in red) was used to stage the seminiferous tubules. Scale bar, 20 μm.

    Techniques Used: Mouse Assay, Staining, Transgenic Assay, TUNEL Assay

    Sperm abnormalities in Slx/Slxl1 -deficient mice. (A) Bar graph representing the percentage of sperm with sperm head abnormalities in Slx/Slxl1 -deficient mice and control. (B) Examples of sperm head abnormalities observed in Slx/Slxl1 -deficient mice (silver-stained epididymal sperm). Scale bar, 10 μm. (C) Bar graph representing the percentage of sperm with abnormal head-tail connections in epididymal sperm from Slx/Slxl1 -deficient mice and control. (D) Examples of head-tail connection anomalies in Slx/Slxl1 -deficient spermatozoa. Scale bar, 10 μm. (E–J) Electron microscopy pictures of WT (E and F) and Slx/Slxl1 -deficient epididymal sperm with partial detachment of the tail (G), parallel alignment of the head and tail (H), curved tail (I), or looped tail (J). Pictures were taken using the same parameters. Scale bar, 1 μm.
    Figure Legend Snippet: Sperm abnormalities in Slx/Slxl1 -deficient mice. (A) Bar graph representing the percentage of sperm with sperm head abnormalities in Slx/Slxl1 -deficient mice and control. (B) Examples of sperm head abnormalities observed in Slx/Slxl1 -deficient mice (silver-stained epididymal sperm). Scale bar, 10 μm. (C) Bar graph representing the percentage of sperm with abnormal head-tail connections in epididymal sperm from Slx/Slxl1 -deficient mice and control. (D) Examples of head-tail connection anomalies in Slx/Slxl1 -deficient spermatozoa. Scale bar, 10 μm. (E–J) Electron microscopy pictures of WT (E and F) and Slx/Slxl1 -deficient epididymal sperm with partial detachment of the tail (G), parallel alignment of the head and tail (H), curved tail (I), or looped tail (J). Pictures were taken using the same parameters. Scale bar, 1 μm.

    Techniques Used: Mouse Assay, Staining, Electron Microscopy

    11) Product Images from "3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy"

    Article Title: 3β-Hydroxysterol Δ24-Reductase on the Surface of Hepatitis C Virus-Related Hepatocellular Carcinoma Cells Can Be a Target for Molecular Targeting Therapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124197

    Identification of the surface DHCR24-recognition domain of 2-152a MAb. ( a ) Alignment of the VH amino acid sequence of 2-152a MAb with the germline configuration. The V, D, J, and CDR sequences were determined by using the IMGT database ( http://www.imgt.org/ ). ( b ) Alignment of the VL amino acid sequence of 2-152a MAb with the germline configuration. The V, J, and CDR sequences were determined by using the IMGT database.
    Figure Legend Snippet: Identification of the surface DHCR24-recognition domain of 2-152a MAb. ( a ) Alignment of the VH amino acid sequence of 2-152a MAb with the germline configuration. The V, D, J, and CDR sequences were determined by using the IMGT database ( http://www.imgt.org/ ). ( b ) Alignment of the VL amino acid sequence of 2-152a MAb with the germline configuration. The V, J, and CDR sequences were determined by using the IMGT database.

    Techniques Used: Sequencing

    Specific uptake mediated by cell-surface DHCR24. ( a ) HCC cell lines (HuH-7, Hep3B, and PLC/PRF/5) and HeLa cells were incubated with 2-152a MAb at 4°C (a temperature that inhibits endocytosis) or 37°C (physiological temperature) for 2 h, and then incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. ( b ) HuH-7 cells were seeded at a density of 5 × 10 3 cells/well in 96-well tissue culture plates. After incubation for 24 h, serial dilutions of 2-152a MAb or mouse IgG were added in the presence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was then assessed using the BrdU ELISA assay kit. Average viability was calculated relative to the viability of untreated cells, which was set at 100%. ( c ) HeLa, Hep3B, and PLC/PRF/5 cells were treated with 2-152a MAb or mouse IgG (10 μg/mL) in the presence or absence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was determined using a BrdU ELISA assay kit. Percent viability was calculated relative to the viability of untreated cells, which was set at 100%. *, p
    Figure Legend Snippet: Specific uptake mediated by cell-surface DHCR24. ( a ) HCC cell lines (HuH-7, Hep3B, and PLC/PRF/5) and HeLa cells were incubated with 2-152a MAb at 4°C (a temperature that inhibits endocytosis) or 37°C (physiological temperature) for 2 h, and then incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. ( b ) HuH-7 cells were seeded at a density of 5 × 10 3 cells/well in 96-well tissue culture plates. After incubation for 24 h, serial dilutions of 2-152a MAb or mouse IgG were added in the presence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was then assessed using the BrdU ELISA assay kit. Average viability was calculated relative to the viability of untreated cells, which was set at 100%. ( c ) HeLa, Hep3B, and PLC/PRF/5 cells were treated with 2-152a MAb or mouse IgG (10 μg/mL) in the presence or absence of saporin-conjugated anti-mouse IgG (1 μg/mL). After 72 h, cell viability was determined using a BrdU ELISA assay kit. Percent viability was calculated relative to the viability of untreated cells, which was set at 100%. *, p

    Techniques Used: Planar Chromatography, Incubation, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Overexpression of DHCR24 on the cell surface of HCC cell lines was decreased by treatment with U18666A and cyclosporin A. ( a ) Cell lysates of each cell line (containing 50 μg of protein) were separated by 10% SDS-PAGE and analyzed by western blotting with 2-152a MAb and an anti-actin MAb. Normal hepatic cell lines: NKNT, and TTNT. HB-derived cell line: HepG2. HCC-derived cell lines: HuH-7, Hep3B, and PLC/PRF/5. ( b ) HuH-7 cells were treated with U18666A (final concentration, 1 μM) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry. ( c ) HuH-7 cells were treated with cyclosporin A (final concentration, 5 or 10 μM) or solvent (cremophor) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry.
    Figure Legend Snippet: Overexpression of DHCR24 on the cell surface of HCC cell lines was decreased by treatment with U18666A and cyclosporin A. ( a ) Cell lysates of each cell line (containing 50 μg of protein) were separated by 10% SDS-PAGE and analyzed by western blotting with 2-152a MAb and an anti-actin MAb. Normal hepatic cell lines: NKNT, and TTNT. HB-derived cell line: HepG2. HCC-derived cell lines: HuH-7, Hep3B, and PLC/PRF/5. ( b ) HuH-7 cells were treated with U18666A (final concentration, 1 μM) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry. ( c ) HuH-7 cells were treated with cyclosporin A (final concentration, 5 or 10 μM) or solvent (cremophor) for 48 h, and then the surface expression of DHCR24 was analyzed by flow cytometry.

    Techniques Used: Over Expression, SDS Page, Western Blot, Derivative Assay, Planar Chromatography, Concentration Assay, Expressing, Flow Cytometry, Cytometry

    152a ChAb can bind to surface DHCR24 and shows anti-HCV activity. ( a ) HuH-7 (1 × 10 6 ) cells were incubated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) at 4°C for 2 h, and then with an Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. ( b ) HuH-7 (1 × 10 6 ) cells were incubated with 152a scFv-hIgG1-Fc at 4°C for 2 h, and then incubated with Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. ( c ) Subgenomic HCV replicon FLR3-1 cells were plated in a 96-well plate at a density of 5 × 10 3 cells/well and allowed to adhere overnight. Then, the supernatant was removed, and the cells were treated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) for 72 h. HCV replication was evaluated by measuring luciferase activity using the Bright-Glo Luciferase Assay System. *, p
    Figure Legend Snippet: 152a ChAb can bind to surface DHCR24 and shows anti-HCV activity. ( a ) HuH-7 (1 × 10 6 ) cells were incubated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) at 4°C for 2 h, and then with an Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. ( b ) HuH-7 (1 × 10 6 ) cells were incubated with 152a scFv-hIgG1-Fc at 4°C for 2 h, and then incubated with Alexa Fluor 488-conjugated goat anti-human IgG at 4°C for 1 h. The cells were then analyzed by flow cytometry. ( c ) Subgenomic HCV replicon FLR3-1 cells were plated in a 96-well plate at a density of 5 × 10 3 cells/well and allowed to adhere overnight. Then, the supernatant was removed, and the cells were treated with the light or heavy chain of the chimeric Ig (152a Ch-L, 152a Ch-H) or 152a ChAb (1 μg/mL, respectively) for 72 h. HCV replication was evaluated by measuring luciferase activity using the Bright-Glo Luciferase Assay System. *, p

    Techniques Used: Activity Assay, Incubation, Flow Cytometry, Cytometry, Luciferase

    12) Product Images from "Enhanced stress tolerance through reduction of G3BP and suppression of stress granules"

    Article Title: Enhanced stress tolerance through reduction of G3BP and suppression of stress granules

    Journal: bioRxiv

    doi: 10.1101/2020.02.03.925677

    MAGE-B2 and DDX5 have opposing roles in the regulation of G3BP1 translation. (A) MAGE-B2 interacts with G3BP1 mRNA by CLIP-qPCR. HEK293 cells stably expressing vector control or TAP-MAGE-B2 were subjected to 150 mJ/cm 2 of UVC (254 nm) before immunoprecipitation of TAP-vector or TAP-MAGE-B2. G3BP1 mRNA or RPLP0 mRNA as a normalization control were then detected by RT-qPCR (n = 3). Relative amount of G3BP1 mRNA in MAGE-B2 pulldown relative to control pulldown after RPLP0 normalization is shown. P value was determined by t test. (B) MAGE-B2 suppresses DDX5 interaction with G3BP1 mRNA. CLIP-qPCR was performed as described above using HEK293 cells stably expressing vector control or TAP-MAGE-B2 to measure enrichment of G3BP1 mRNA after DDX5 pulldown (n = 3). Relative levels of G3BP1 mRNA in DDX5 pulldown relative to IgG control after RPLP0 normalization is shown. P value was determined by t test. (C) Loss of MAGE-B2 enhances DDX5 interaction with G3BP1 mRNA. CLIP-qPCR was performed on WT or MAGE-B2 KO U2OS cells to measure enrichment of G3BP1 mRNA for DDX5 (n = 3) as described above. P value was determined by t test. (D) MAGE-B2 and DDX5 compete for binding to G3BP1 mRNA. Biotinylated control ( Luc CDS) or bait ( Luc CDS with G3BP1 5’ and 3’ UTRs) transcripts were pulled down from either WT or MAGE-B2 KO U2OS cells and subjected to immunoblotting for DDX5, MAGE-B2 or TRIM28 as a negative control. (E and F) DDX5 enhances G3BP1 translation in vitro . In vitro translation assays were performed using rabbit reticulocyte lysate with increasing amounts of recombinant GST (control), DDX5, or MAGE-B2 to measure translation of a luciferase reporter (n = 3) containing G3BP1 5’ and 3’ UTRs (E), or only G3BP1 5’ UTR (F). P values were determined by t test. (G and H) DDX5 helicase activity is required for enhancing G3BP1 translation in vitro . In vitro translation assays were performed using increasing amounts of recombinant GST (control), DDX5 WT, or DDX5 K144N (helicase dead mutant) to measure translation of a luciferase reporter (n = 3) containing G3BP1 5’ UTR (G) or G3BP1 5’ UTR lacking the DDX5 binding motif (10 nucleotide deletion) (H). P values were determined by t test. (I) In vitro translation assays reveal competition between DDX5 and MAGE-B2 for G3BP1 regulation via the 5’ UTR . In vitro translation assays were performed using recombinant DDX5 and titrating increasing amounts of GST (control) or MAGE-B2 to measure translation (n = 3) of a luciferase reporter containing G3BP1 5’ UTR. P values were determined by t test. (J) Regulation of G3BP1 translation via the 5’ UTR. Basal translation of the various luciferase reporters relative to control (Luc CDS) reveals that the presence of G3BP1 5’ UTR suppresses translation (n = 3). P values were determined by one-way ANOVA. (K) Model of the mechanism by which MAGE-B2 reduces G3BP and suppresses SG. MAGE-B2 inhibits G3BP translation by competing with the translational activator DDX5. This results in reduced G3BP protein levels, suppression of SG, and increased cellular stress threshold. Data are mean ± SD. Asterisks indicate significant differences from the control (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, n.s. = not significant) See also Figure S4.
    Figure Legend Snippet: MAGE-B2 and DDX5 have opposing roles in the regulation of G3BP1 translation. (A) MAGE-B2 interacts with G3BP1 mRNA by CLIP-qPCR. HEK293 cells stably expressing vector control or TAP-MAGE-B2 were subjected to 150 mJ/cm 2 of UVC (254 nm) before immunoprecipitation of TAP-vector or TAP-MAGE-B2. G3BP1 mRNA or RPLP0 mRNA as a normalization control were then detected by RT-qPCR (n = 3). Relative amount of G3BP1 mRNA in MAGE-B2 pulldown relative to control pulldown after RPLP0 normalization is shown. P value was determined by t test. (B) MAGE-B2 suppresses DDX5 interaction with G3BP1 mRNA. CLIP-qPCR was performed as described above using HEK293 cells stably expressing vector control or TAP-MAGE-B2 to measure enrichment of G3BP1 mRNA after DDX5 pulldown (n = 3). Relative levels of G3BP1 mRNA in DDX5 pulldown relative to IgG control after RPLP0 normalization is shown. P value was determined by t test. (C) Loss of MAGE-B2 enhances DDX5 interaction with G3BP1 mRNA. CLIP-qPCR was performed on WT or MAGE-B2 KO U2OS cells to measure enrichment of G3BP1 mRNA for DDX5 (n = 3) as described above. P value was determined by t test. (D) MAGE-B2 and DDX5 compete for binding to G3BP1 mRNA. Biotinylated control ( Luc CDS) or bait ( Luc CDS with G3BP1 5’ and 3’ UTRs) transcripts were pulled down from either WT or MAGE-B2 KO U2OS cells and subjected to immunoblotting for DDX5, MAGE-B2 or TRIM28 as a negative control. (E and F) DDX5 enhances G3BP1 translation in vitro . In vitro translation assays were performed using rabbit reticulocyte lysate with increasing amounts of recombinant GST (control), DDX5, or MAGE-B2 to measure translation of a luciferase reporter (n = 3) containing G3BP1 5’ and 3’ UTRs (E), or only G3BP1 5’ UTR (F). P values were determined by t test. (G and H) DDX5 helicase activity is required for enhancing G3BP1 translation in vitro . In vitro translation assays were performed using increasing amounts of recombinant GST (control), DDX5 WT, or DDX5 K144N (helicase dead mutant) to measure translation of a luciferase reporter (n = 3) containing G3BP1 5’ UTR (G) or G3BP1 5’ UTR lacking the DDX5 binding motif (10 nucleotide deletion) (H). P values were determined by t test. (I) In vitro translation assays reveal competition between DDX5 and MAGE-B2 for G3BP1 regulation via the 5’ UTR . In vitro translation assays were performed using recombinant DDX5 and titrating increasing amounts of GST (control) or MAGE-B2 to measure translation (n = 3) of a luciferase reporter containing G3BP1 5’ UTR. P values were determined by t test. (J) Regulation of G3BP1 translation via the 5’ UTR. Basal translation of the various luciferase reporters relative to control (Luc CDS) reveals that the presence of G3BP1 5’ UTR suppresses translation (n = 3). P values were determined by one-way ANOVA. (K) Model of the mechanism by which MAGE-B2 reduces G3BP and suppresses SG. MAGE-B2 inhibits G3BP translation by competing with the translational activator DDX5. This results in reduced G3BP protein levels, suppression of SG, and increased cellular stress threshold. Data are mean ± SD. Asterisks indicate significant differences from the control (* = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, n.s. = not significant) See also Figure S4.

    Techniques Used: Cross-linking Immunoprecipitation, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Negative Control, In Vitro, Recombinant, Luciferase, Activity Assay, Mutagenesis

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

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

    Journal: Histochemistry and Cell Biology

    doi: 10.1007/s00418-014-1199-9

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

    Techniques Used: Fluorescence, Staining

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

    Techniques Used: Western Blot, Transfection

    14) Product Images from "Fyn Tyrosine Kinase Increases Apolipoprotein E Receptor 2 Levels and Phosphorylation"

    Article Title: Fyn Tyrosine Kinase Increases Apolipoprotein E Receptor 2 Levels and Phosphorylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0110845

    ApoER2 is phosphorylated in vivo . A. Cortical (CTX) and hippocampal (HPC) lysates from wild-type mice were collected on postnatal days 1, 5, and 15, homogenized in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, and analyzed for ApoER2. Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Fyn, and Tubulin. B. Levels of total ApoER2 were plotted from Western blots in A. # p
    Figure Legend Snippet: ApoER2 is phosphorylated in vivo . A. Cortical (CTX) and hippocampal (HPC) lysates from wild-type mice were collected on postnatal days 1, 5, and 15, homogenized in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, and analyzed for ApoER2. Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Fyn, and Tubulin. B. Levels of total ApoER2 were plotted from Western blots in A. # p

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

    Fyn phosphorylates Dab1 first at the ab site. A. COS7 cells were transfected with Fyn, Dab1-WT, or Fyn and Dab1-WT together. Lysates were immunoprecipitated (IP, top) with either 4G10 or mouse IgG and probed for Dab1. Input refers to the starting material that was not subjected to IP. Western blots (bottom) show total levels of phosphorylated proteins (4G10), Dab1, Fyn, and β–actin. The Dab1 band is indicated (arrow). B. Schematic of Dab1 constructs. WT: Wild-type, PTB: phosphotyrosine binding domain. C. COS7 cells were transfected with Dab1-WT, Dab1-ab, or Dab1-cd and either empty vector or Fyn. Blots were probed for phosphorylated proteins, and the presumed Dab1 and Fyn bands are indicated (arrows).
    Figure Legend Snippet: Fyn phosphorylates Dab1 first at the ab site. A. COS7 cells were transfected with Fyn, Dab1-WT, or Fyn and Dab1-WT together. Lysates were immunoprecipitated (IP, top) with either 4G10 or mouse IgG and probed for Dab1. Input refers to the starting material that was not subjected to IP. Western blots (bottom) show total levels of phosphorylated proteins (4G10), Dab1, Fyn, and β–actin. The Dab1 band is indicated (arrow). B. Schematic of Dab1 constructs. WT: Wild-type, PTB: phosphotyrosine binding domain. C. COS7 cells were transfected with Dab1-WT, Dab1-ab, or Dab1-cd and either empty vector or Fyn. Blots were probed for phosphorylated proteins, and the presumed Dab1 and Fyn bands are indicated (arrows).

    Techniques Used: Transfection, Immunoprecipitation, Western Blot, Construct, Binding Assay, Plasmid Preparation

    Fyn phosphorylates ApoER2. A. COS7 cells were co-transfected with untagged ApoER2 and Fyn or empty vector. Lysates were collected in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, and analyzed for ApoER2. Input refers to the starting material that was not subjected to IP. Lane numbers are indicated for clarity. B. Schematic of WT ApoER2 and the mutant with all three intracellular tyrosines mutated to phenylalanines (tYF). The signal peptide (SP), N-terminal HA tag, and C-terminal myc tag are indicated. C. COS7 cells were co-transfected with different ApoER2 mutants (2 single mutants on left, 1 single mutant and triple mutant on right) and Fyn or empty vector and immunoprecipitated as in A, and analyzed for myc. Below are Western blots showing total levels of ApoER2, Fyn, and β–actin. D. Quantification of the upper-phosphorylated ApoER2 band (indicated by arrow in boxed example) over total ApoER2 from C. ** p
    Figure Legend Snippet: Fyn phosphorylates ApoER2. A. COS7 cells were co-transfected with untagged ApoER2 and Fyn or empty vector. Lysates were collected in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, and analyzed for ApoER2. Input refers to the starting material that was not subjected to IP. Lane numbers are indicated for clarity. B. Schematic of WT ApoER2 and the mutant with all three intracellular tyrosines mutated to phenylalanines (tYF). The signal peptide (SP), N-terminal HA tag, and C-terminal myc tag are indicated. C. COS7 cells were co-transfected with different ApoER2 mutants (2 single mutants on left, 1 single mutant and triple mutant on right) and Fyn or empty vector and immunoprecipitated as in A, and analyzed for myc. Below are Western blots showing total levels of ApoER2, Fyn, and β–actin. D. Quantification of the upper-phosphorylated ApoER2 band (indicated by arrow in boxed example) over total ApoER2 from C. ** p

    Techniques Used: Transfection, Plasmid Preparation, Immunoprecipitation, Mutagenesis, Western Blot

    ApoER2 levels increase independently of the intracellular domain of ApoER2. A. Schematic of tagged ApoER2 constructs. Wild-type (WT) murine ApoER2 with both an N-terminal HA tag and a C-terminal myc tag. ΔICD is missing amino acids 731–841 and the myc tag. SP: Signal peptide. B. COS7 cells were co-transfected with either ApoER2 WT or ΔICD and either Fyn or empty vector. Lysates were collected in RIPA buffer and analyzed for HA, Fyn, and Tubulin. * p
    Figure Legend Snippet: ApoER2 levels increase independently of the intracellular domain of ApoER2. A. Schematic of tagged ApoER2 constructs. Wild-type (WT) murine ApoER2 with both an N-terminal HA tag and a C-terminal myc tag. ΔICD is missing amino acids 731–841 and the myc tag. SP: Signal peptide. B. COS7 cells were co-transfected with either ApoER2 WT or ΔICD and either Fyn or empty vector. Lysates were collected in RIPA buffer and analyzed for HA, Fyn, and Tubulin. * p

    Techniques Used: Construct, Transfection, Plasmid Preparation

    Fyn phosphorylation of ApoER2 is increased in the presence of Dab1. A. COS7 cells were transfected with untagged ApoER2 and either empty vector, Fyn, Dab1-WT, or Fyn and Dab1-WT together. Lysates were collected in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, run on a Western blot, and probed for ApoER2. Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Dab1, Fyn, and β–actin. B–C. COS7 cells were transfected and analyzed as in A, but with Dab1-ab (B) or Dab1-cd (C) instead of Dab1-WT.
    Figure Legend Snippet: Fyn phosphorylation of ApoER2 is increased in the presence of Dab1. A. COS7 cells were transfected with untagged ApoER2 and either empty vector, Fyn, Dab1-WT, or Fyn and Dab1-WT together. Lysates were collected in IP buffer and immunoprecipitated (IP) with either 4G10 or mouse IgG, run on a Western blot, and probed for ApoER2. Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Dab1, Fyn, and β–actin. B–C. COS7 cells were transfected and analyzed as in A, but with Dab1-ab (B) or Dab1-cd (C) instead of Dab1-WT.

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

    Fyn increases ApoER2 levels. A. Brains from 3 week old wild-type (WT) and Fyn knock-out (KO) mice were homogenized in RIPA buffer and analyzed for ApoER2, Fyn, or β-tubulin by immunoblot. ApoER2 levels were normalized to β-tubulin. * p
    Figure Legend Snippet: Fyn increases ApoER2 levels. A. Brains from 3 week old wild-type (WT) and Fyn knock-out (KO) mice were homogenized in RIPA buffer and analyzed for ApoER2, Fyn, or β-tubulin by immunoblot. ApoER2 levels were normalized to β-tubulin. * p

    Techniques Used: Knock-Out, Mouse Assay

    Fyn’s effects on ApoER2 levels depend on Fyn’s kinase activity. A. COS7 cells were co-transfected with ApoER2 and either Fyn or empty vector and treated with PP2 or DMSO (control) for 6 hours. Lysates were collected in RIPA buffer, Western blotted and probed for ApoER2, Fyn, and tubulin. ** p
    Figure Legend Snippet: Fyn’s effects on ApoER2 levels depend on Fyn’s kinase activity. A. COS7 cells were co-transfected with ApoER2 and either Fyn or empty vector and treated with PP2 or DMSO (control) for 6 hours. Lysates were collected in RIPA buffer, Western blotted and probed for ApoER2, Fyn, and tubulin. ** p

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Western Blot

    Fyn increases the interaction between ApoER2 and Dab1. COS7 cells were co-transfected with double-tagged ApoER2 and either Fyn, Dab1, or Fyn and Dab1 and lysates collected in IP buffer after 20 hours. A. Lysates were immunoprecipitated (IP) with either GFP (for Dab1) or rabbit IgG, run on a Western blot, and probed for HA (for ApoER2). Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Dab1, Fyn, and Tubulin. B. The lysates used in A were precipitated with either HA (for ApoER2) or mouse IgG, and analyzed for Dab1-GFP. C. Quantification of lanes 2 and 3 from A. * p
    Figure Legend Snippet: Fyn increases the interaction between ApoER2 and Dab1. COS7 cells were co-transfected with double-tagged ApoER2 and either Fyn, Dab1, or Fyn and Dab1 and lysates collected in IP buffer after 20 hours. A. Lysates were immunoprecipitated (IP) with either GFP (for Dab1) or rabbit IgG, run on a Western blot, and probed for HA (for ApoER2). Input refers to the starting material that was not subjected to IP. Below are Western blots showing total levels of ApoER2, Dab1, Fyn, and Tubulin. B. The lysates used in A were precipitated with either HA (for ApoER2) or mouse IgG, and analyzed for Dab1-GFP. C. Quantification of lanes 2 and 3 from A. * p

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    15) Product Images from "Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements"

    Article Title: Androgen-induced TOP2B mediated double strand breaks and prostate cancer gene rearrangements

    Journal: Nature genetics

    doi: 10.1038/ng.613

    Androgen-induced TOP2B mediated DSB are recombinogenic and promote de novo production of TMPRSS2-ERG fusion genes. a , Selection of TMPRSS2 regions showing high (Intron 1-T8) and low ( TMPRSS2 -Exon 6) KSDS enrichment in response to DHT stimulation of LAPC4 cells. b , DHT-induced breaks can be detected in plasmids containing sequences surrounding region T8 of TMPRSS2 intron 1 (pcDNA6.2-IN-1) as evidenced by increased biotin labeling in DHT stimulated LAPC4 cells transfected with this plasmid. Plasmids containing TMPRSS2 exon 6 (pcDNA6.2-EX-6) served as a negative control. c , Schematic of androgen-induced genomic recombination assay in LAPC4 cells transfected with pcDNA6.2-IN-1 or pcDNA6.2-EX-6, which contain a blasticidin resistance gene. Number of colonies represents the number of recombination events allowing integration of the blasticidin resistance vectors into the LAPC4 genome. d , Representative results of genomic recombination assays. e , pcDNA6.2-IN-1 transfected LAPC4 cells produced significantly more androgen induced recombination events than pcDNA6.2-EX-6 transfected cells. Treatment with sh-TOP2B abolished this effect. f , While both showed similar recombination frequency at the vector backbone, recombination frequency within the IN-1 insert was significantly higher than that in the EX-6 insert in pooled colonies as determined using the strategy shown on the right. Data are shown as mean ± SE of two to three replicates. g , DHT-stimulation of LAPC4 cells leads to increased TMPRSS2-ERG fusion transcripts compared to background levels in LAPC4 cells grown in androgen-containing (steady-state) or androgen-deprived (control) media. Pharmacological or genetic modulation of TOP2B (Mer, sh-TOP2B), PARP1 (3-AB, PJ-34, si-PARP1), or DNA-PK CS (Wort, si-DNA-PK CS ), reduces TMPRSS2-ERG fusion transcripts without significantly altering GAPDH or TBP expression. h , DHT-stimulation leads to de novo formation of TMPRSS2-ERG fusion transcripts in LNCaP cells.
    Figure Legend Snippet: Androgen-induced TOP2B mediated DSB are recombinogenic and promote de novo production of TMPRSS2-ERG fusion genes. a , Selection of TMPRSS2 regions showing high (Intron 1-T8) and low ( TMPRSS2 -Exon 6) KSDS enrichment in response to DHT stimulation of LAPC4 cells. b , DHT-induced breaks can be detected in plasmids containing sequences surrounding region T8 of TMPRSS2 intron 1 (pcDNA6.2-IN-1) as evidenced by increased biotin labeling in DHT stimulated LAPC4 cells transfected with this plasmid. Plasmids containing TMPRSS2 exon 6 (pcDNA6.2-EX-6) served as a negative control. c , Schematic of androgen-induced genomic recombination assay in LAPC4 cells transfected with pcDNA6.2-IN-1 or pcDNA6.2-EX-6, which contain a blasticidin resistance gene. Number of colonies represents the number of recombination events allowing integration of the blasticidin resistance vectors into the LAPC4 genome. d , Representative results of genomic recombination assays. e , pcDNA6.2-IN-1 transfected LAPC4 cells produced significantly more androgen induced recombination events than pcDNA6.2-EX-6 transfected cells. Treatment with sh-TOP2B abolished this effect. f , While both showed similar recombination frequency at the vector backbone, recombination frequency within the IN-1 insert was significantly higher than that in the EX-6 insert in pooled colonies as determined using the strategy shown on the right. Data are shown as mean ± SE of two to three replicates. g , DHT-stimulation of LAPC4 cells leads to increased TMPRSS2-ERG fusion transcripts compared to background levels in LAPC4 cells grown in androgen-containing (steady-state) or androgen-deprived (control) media. Pharmacological or genetic modulation of TOP2B (Mer, sh-TOP2B), PARP1 (3-AB, PJ-34, si-PARP1), or DNA-PK CS (Wort, si-DNA-PK CS ), reduces TMPRSS2-ERG fusion transcripts without significantly altering GAPDH or TBP expression. h , DHT-stimulation leads to de novo formation of TMPRSS2-ERG fusion transcripts in LNCaP cells.

    Techniques Used: Selection, Labeling, Transfection, Plasmid Preparation, Negative Control, Recombination Assay, Produced, Expressing

    16) Product Images from ""

    Article Title:

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.084590

    Physical interaction of CRIF1 and NRF2. A, exogenous NRF2 and CRIF1 interact under normal reducing conditions. Total lysates of HEK293 cells transiently co-transfected for 24 h with expression vectors for FLAG-NRF2 and GFP-CRIF1, either individually or together, were immunoprecipitated with anti-GFP or anti-FLAG antibodies and then used for WB analysis with anti-FLAG and anti-GFP antibodies as indicated. B, exogenous NRF2 and CRIF1 also interact under oxidative stress conditions. HEK293 cells co-transfected with GFP-NRF2 and FLAG-CRIF1 were then incubated with t -BHQ (100 μ m ) for the times indicated and analyzed as in A. C and D, endogenous CRIF1 interacts with exogenous NRF2 in the presence of t -BHQ or SFN. MCF-7 cells transfected with GFP as a negative control and GFP-NRF2 expression vector for 24 h and then treated with either 100 μ m t -BHQ ( C ) or 5 μ m SFN ( D ) for the times indicated. IP-WBs were analyzed as in A except that an anti-CRIF1 mouse antibody was used. One-tenth of the total cell lysates was also used for WB analysis ( bottom panels ). For the top panels of C and D , IP-WB images were scanned, and quantified results are shown as bar graphs ( below the IP-WB images). We quantified the slower migrating form of CRIF1, which is the form that predominantly interacts with NRF2, and the results are shown in the bar graphs . The results were from triplicate experiments, and a representative image of IP-WB is shown.
    Figure Legend Snippet: Physical interaction of CRIF1 and NRF2. A, exogenous NRF2 and CRIF1 interact under normal reducing conditions. Total lysates of HEK293 cells transiently co-transfected for 24 h with expression vectors for FLAG-NRF2 and GFP-CRIF1, either individually or together, were immunoprecipitated with anti-GFP or anti-FLAG antibodies and then used for WB analysis with anti-FLAG and anti-GFP antibodies as indicated. B, exogenous NRF2 and CRIF1 also interact under oxidative stress conditions. HEK293 cells co-transfected with GFP-NRF2 and FLAG-CRIF1 were then incubated with t -BHQ (100 μ m ) for the times indicated and analyzed as in A. C and D, endogenous CRIF1 interacts with exogenous NRF2 in the presence of t -BHQ or SFN. MCF-7 cells transfected with GFP as a negative control and GFP-NRF2 expression vector for 24 h and then treated with either 100 μ m t -BHQ ( C ) or 5 μ m SFN ( D ) for the times indicated. IP-WBs were analyzed as in A except that an anti-CRIF1 mouse antibody was used. One-tenth of the total cell lysates was also used for WB analysis ( bottom panels ). For the top panels of C and D , IP-WB images were scanned, and quantified results are shown as bar graphs ( below the IP-WB images). We quantified the slower migrating form of CRIF1, which is the form that predominantly interacts with NRF2, and the results are shown in the bar graphs . The results were from triplicate experiments, and a representative image of IP-WB is shown.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Western Blot, Incubation, Negative Control, Plasmid Preparation

    CRIF1 promotes NRF2 ubiquitination. A, total lysates of COS-1 cells co-transfected for 24 h with the expression vector combinations (encoding full-length NRF2) indicated at the top of the figure were divided and then analyzed either directly on WBs (1/10 of the lysates) or first immunoprecipitated with anti-FLAG antibody and then used for standard WB analysis with the antibodies indicated in the right-hand margin. B, schematic showing the amino acid composition of the N- and C-terminal lysine clusters and their locations relative to previously described NRF2 landmarks. C, cells (COS-1) co-transfected for 24 h with indicated expression vectors, including NRF2 mutants ( mt ) with four lysine to arginine residues, were subjected to IP and WB analysis as described in A. wt , wild type. D, COS-1 cells were co-transfected for 24 h with the indicated expression vectors. The NRF2 deletion mutant encoding expression vectors encoded two tags, His and V5, and CRIF1 was in pCDNA and ubiquitin was tagged with HA. Total lysates were immunoprecipitated with the anti-His (NRF2) antibody and analyzed as in A and C with the antibodies indicated in the right-hand margin . (Ub) n NRF2 indicates polyubiquitinated forms of NRF2.
    Figure Legend Snippet: CRIF1 promotes NRF2 ubiquitination. A, total lysates of COS-1 cells co-transfected for 24 h with the expression vector combinations (encoding full-length NRF2) indicated at the top of the figure were divided and then analyzed either directly on WBs (1/10 of the lysates) or first immunoprecipitated with anti-FLAG antibody and then used for standard WB analysis with the antibodies indicated in the right-hand margin. B, schematic showing the amino acid composition of the N- and C-terminal lysine clusters and their locations relative to previously described NRF2 landmarks. C, cells (COS-1) co-transfected for 24 h with indicated expression vectors, including NRF2 mutants ( mt ) with four lysine to arginine residues, were subjected to IP and WB analysis as described in A. wt , wild type. D, COS-1 cells were co-transfected for 24 h with the indicated expression vectors. The NRF2 deletion mutant encoding expression vectors encoded two tags, His and V5, and CRIF1 was in pCDNA and ubiquitin was tagged with HA. Total lysates were immunoprecipitated with the anti-His (NRF2) antibody and analyzed as in A and C with the antibodies indicated in the right-hand margin . (Ub) n NRF2 indicates polyubiquitinated forms of NRF2.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, Western Blot, Mutagenesis

    A proposed model for CRIF1 and NRF2 interaction. This schematic drawing demonstrates how CRIF1 regulates NRF2 ubiquitination and its transcriptional regulation in either reduced or oxidized conditions. We found two forms of CRIF1, a fast migrating form in the cytosol and a slow migrating form in the nucleus. Because nuclear CRIF1 is the predominant form that interacts with NRF2, this model is focused on role of nuclear CRIF1. In reduced conditions, CRIF1 binds and ubiquitinates nuclear NRF2, which limits its ability to function as a transcription factor. In oxidized conditions, NRF2 bound by KEAP1 will escape from proteasome-mediated degradation and will enter into the nucleus. Perhaps excess amounts of NRF2, which cannot bind ARE-containing promoters or enhancers, may bind nuclear CRIF1 and undergo post-translational modifications. Whether these post-translational modifications of NRF2 occur in the cytosol or nucleus needs further investigation. To include the faster migrating form of CRIF1 (cytosolic), which weakly interacts with NRF2 in redox-independent manner into this model, we need to know the following: 1) whether KEAP1 and the fast migrating form of CRIF1 are in the same cellular compartment; 2) whether KEAP1 and CRIF1 compete with each other to bind N-terminal of NRF2; and 3) a precise DNA sequence of the fast migrating form of CRIF1.
    Figure Legend Snippet: A proposed model for CRIF1 and NRF2 interaction. This schematic drawing demonstrates how CRIF1 regulates NRF2 ubiquitination and its transcriptional regulation in either reduced or oxidized conditions. We found two forms of CRIF1, a fast migrating form in the cytosol and a slow migrating form in the nucleus. Because nuclear CRIF1 is the predominant form that interacts with NRF2, this model is focused on role of nuclear CRIF1. In reduced conditions, CRIF1 binds and ubiquitinates nuclear NRF2, which limits its ability to function as a transcription factor. In oxidized conditions, NRF2 bound by KEAP1 will escape from proteasome-mediated degradation and will enter into the nucleus. Perhaps excess amounts of NRF2, which cannot bind ARE-containing promoters or enhancers, may bind nuclear CRIF1 and undergo post-translational modifications. Whether these post-translational modifications of NRF2 occur in the cytosol or nucleus needs further investigation. To include the faster migrating form of CRIF1 (cytosolic), which weakly interacts with NRF2 in redox-independent manner into this model, we need to know the following: 1) whether KEAP1 and the fast migrating form of CRIF1 are in the same cellular compartment; 2) whether KEAP1 and CRIF1 compete with each other to bind N-terminal of NRF2; and 3) a precise DNA sequence of the fast migrating form of CRIF1.

    Techniques Used: Sequencing

    CRIF1-driven NRF2 ubiquitination and degradation are redox-independent. Total lysates of MCF-7 cells co-transfected with the indicated expression vectors for 24 h were treated with either 100 μ m of t -BHQ or 5 μ m of SFN for 6 h and were immunoprecipitated with an anti-His antibody (for NRF2) followed by analysis on WBs with anti-HA antibody (for ubiquitin). One-tenth of the total cell lysates was used to determine the expression level of each transfected DNA plasmid by WB analysis. Anti-V5 antibody was used to detect exogenously expressed NRF2, and the anti-FLAG antibody was used to detect either CRIF1 or KEAP1. The two proteins, FLAG-CRIF1 and FLAG-KEAP1, were identified by their molecular weights. FLAG-CRIF1 and FLAG-KEAP1 were detected at ∼30 and 70 kDa, respectively.
    Figure Legend Snippet: CRIF1-driven NRF2 ubiquitination and degradation are redox-independent. Total lysates of MCF-7 cells co-transfected with the indicated expression vectors for 24 h were treated with either 100 μ m of t -BHQ or 5 μ m of SFN for 6 h and were immunoprecipitated with an anti-His antibody (for NRF2) followed by analysis on WBs with anti-HA antibody (for ubiquitin). One-tenth of the total cell lysates was used to determine the expression level of each transfected DNA plasmid by WB analysis. Anti-V5 antibody was used to detect exogenously expressed NRF2, and the anti-FLAG antibody was used to detect either CRIF1 or KEAP1. The two proteins, FLAG-CRIF1 and FLAG-KEAP1, were identified by their molecular weights. FLAG-CRIF1 and FLAG-KEAP1 were detected at ∼30 and 70 kDa, respectively.

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

    CRIF1 interacts with both N- and C-terminal sequences of NRF2. A, IVT CRIF1 and FLAG-NRF2 proteins interact. 35 S-Labeled proteins (see “Experimental Procedures”) were mixed, immunoprecipitated with an anti-FLAG antibody, subjected to SDS-PAGE, and exposed to x-ray film. These experiments were repeated three times and yielded similar results; a representative result is shown. 35 S-Labeled IVT luciferase was used as the negative control. B, schematic of the NRF2 deletion mutants used for the experiments in C. C, CRIF1 interacts with both N- and C-terminal regions of NRF2. Mixtures of unlabeled IVT full-length FLAG-CRIF1 and three 35 S-labeled NRF2 deletion mutants were immunoprecipitated with the anti-FLAG antibody, subjected to SDS-PAGE, and exposed to x-ray film.
    Figure Legend Snippet: CRIF1 interacts with both N- and C-terminal sequences of NRF2. A, IVT CRIF1 and FLAG-NRF2 proteins interact. 35 S-Labeled proteins (see “Experimental Procedures”) were mixed, immunoprecipitated with an anti-FLAG antibody, subjected to SDS-PAGE, and exposed to x-ray film. These experiments were repeated three times and yielded similar results; a representative result is shown. 35 S-Labeled IVT luciferase was used as the negative control. B, schematic of the NRF2 deletion mutants used for the experiments in C. C, CRIF1 interacts with both N- and C-terminal regions of NRF2. Mixtures of unlabeled IVT full-length FLAG-CRIF1 and three 35 S-labeled NRF2 deletion mutants were immunoprecipitated with the anti-FLAG antibody, subjected to SDS-PAGE, and exposed to x-ray film.

    Techniques Used: Labeling, Immunoprecipitation, SDS Page, Luciferase, Negative Control

    Knockdown of CRIF1 on NRF2-, t -BHQ-, or SFN-induced gene expression. A, endogenous CRIF1 levels limit the ability of the HO-1 reporter plasmid to respond to inducing treatments. Cells pretreated with siRNA (control, CRIF1–263, or -379) for 48 h were transfected with the DNA plasmid as indicated for 16 h and harvested for luciferase ( Luc ) analysis ( left-hand bar graph ). Next, cells were transfected similarly as in the left panel (but without NRF2) and then exposed for an additional 24 h to either DMSO or 100 μ m of t -BHQ before measuring the luciferase activity ( right-hand bar graph ). The luciferase activities in all figures were normalized to the value obtained with the empty expression plasmid control, unless otherwise indicated or as described under “Experimental Procedures,” and are presented as mean luciferase activity ± S.E. of n = 4 wells. A representative of three independent experiments that yielded similar results is shown in each panel. * means p
    Figure Legend Snippet: Knockdown of CRIF1 on NRF2-, t -BHQ-, or SFN-induced gene expression. A, endogenous CRIF1 levels limit the ability of the HO-1 reporter plasmid to respond to inducing treatments. Cells pretreated with siRNA (control, CRIF1–263, or -379) for 48 h were transfected with the DNA plasmid as indicated for 16 h and harvested for luciferase ( Luc ) analysis ( left-hand bar graph ). Next, cells were transfected similarly as in the left panel (but without NRF2) and then exposed for an additional 24 h to either DMSO or 100 μ m of t -BHQ before measuring the luciferase activity ( right-hand bar graph ). The luciferase activities in all figures were normalized to the value obtained with the empty expression plasmid control, unless otherwise indicated or as described under “Experimental Procedures,” and are presented as mean luciferase activity ± S.E. of n = 4 wells. A representative of three independent experiments that yielded similar results is shown in each panel. * means p

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Luciferase, Activity Assay

    CRIF1 regulates ROS accumulation and cell sensitivity to oxidative stress. A, CRIF1 overexpression increases ROS accumulation. ROS accumulation in MCF-7 cells transfected for 24 h and then treated with various doses of H 2 O 2 for 24 h was measured as described under “Experimental Procedures.” B, CRIF1 knockdown decreases ROS accumulation. ROS levels in MCF-7 cells transfected for 48 h and then treated with H 2 O 2 for 24 h were measured as in A. C–F, CRIF1 affects the ability of the cell to survive oxidative stress. C and D, MCF-7 cells transfected with GFP-CRIF1 or FLAG-CRIF1 ( versus empty vectors, pEGFP-N1 or pCMV-Tag) for 24 h and then treated with H 2 O 2 for 24 h ( C ) or paraquat for 72 h ( D ) were assayed for viability by standard MTT assays. Values are presented as means ± S.E. of cell viability in 10 replicate wells relative to the untreated controls (no DNA or empty vector and no killing agents). The results shown are representative of three independent experiments. E and F, cells (MCF-7) pretreated with the indicated siRNAs (control versus CRIF1–263 or -379) for 48 h were treated with H 2 O 2 for 24 h ( E ) or paraquat for 72 h ( F ) when MTT assays were done.
    Figure Legend Snippet: CRIF1 regulates ROS accumulation and cell sensitivity to oxidative stress. A, CRIF1 overexpression increases ROS accumulation. ROS accumulation in MCF-7 cells transfected for 24 h and then treated with various doses of H 2 O 2 for 24 h was measured as described under “Experimental Procedures.” B, CRIF1 knockdown decreases ROS accumulation. ROS levels in MCF-7 cells transfected for 48 h and then treated with H 2 O 2 for 24 h were measured as in A. C–F, CRIF1 affects the ability of the cell to survive oxidative stress. C and D, MCF-7 cells transfected with GFP-CRIF1 or FLAG-CRIF1 ( versus empty vectors, pEGFP-N1 or pCMV-Tag) for 24 h and then treated with H 2 O 2 for 24 h ( C ) or paraquat for 72 h ( D ) were assayed for viability by standard MTT assays. Values are presented as means ± S.E. of cell viability in 10 replicate wells relative to the untreated controls (no DNA or empty vector and no killing agents). The results shown are representative of three independent experiments. E and F, cells (MCF-7) pretreated with the indicated siRNAs (control versus CRIF1–263 or -379) for 48 h were treated with H 2 O 2 for 24 h ( E ) or paraquat for 72 h ( F ) when MTT assays were done.

    Techniques Used: Over Expression, Transfection, MTT Assay, Plasmid Preparation

    Overexpression of CRIF1 represses NRF2 target gene expression. These experiments used luciferase reporter plasmids that contain AREs from the HO-1 enhancer. A, schematic of the HO-1 gene enhancer region present in the reporter plasmid used in B and C . The two AREs have overlapping 12- O -tetradecanoylphorbol-13-acetate-response elements (TRE or AP-1-binding sites). The M2 mutant contains 2 bp changes in each AP-1 site, leaving the ARE core sequence (TGAC nnn GC) intact. The M239 mutant contains multiple base pair changes that affect both ARE core sequences and the AP-1-binding sites. WT , wild type. B, effects of CRIF1 overexpression on basal and induced expression from wild-type and mutant HO-1 reporter plasmids. Total lysates of MCF-7 cells transfected with various DNA plasmids for 24 h as indicated were measured for luciferase ( Luc ) activity. C, effects of CRIF1 overexpression on t -BHQ-induced HO-1 luciferase reporter. Cells transfected with HO-1 reporter and FLAG-CRIF1 (except NRF2) for 16 h were incubated with either DMSO (control vehicle) or t -BHQ (100 μ m ) for 24 h before harvesting for measuring luciferase activity. D and E, effects of CRIF1 on NRF2 target gene expression. Total RNA was isolated from the MCF-7 cells infected with control Ad-GFP versus Ad-GFP-CRIF1 for 24 h and were treated with 100 μ m of t -BHQ ( D ) or 5 μ m of SFN ( E ). Real time PCR for HO-1 and GCLC was performed as described under “Experimental Procedures.”
    Figure Legend Snippet: Overexpression of CRIF1 represses NRF2 target gene expression. These experiments used luciferase reporter plasmids that contain AREs from the HO-1 enhancer. A, schematic of the HO-1 gene enhancer region present in the reporter plasmid used in B and C . The two AREs have overlapping 12- O -tetradecanoylphorbol-13-acetate-response elements (TRE or AP-1-binding sites). The M2 mutant contains 2 bp changes in each AP-1 site, leaving the ARE core sequence (TGAC nnn GC) intact. The M239 mutant contains multiple base pair changes that affect both ARE core sequences and the AP-1-binding sites. WT , wild type. B, effects of CRIF1 overexpression on basal and induced expression from wild-type and mutant HO-1 reporter plasmids. Total lysates of MCF-7 cells transfected with various DNA plasmids for 24 h as indicated were measured for luciferase ( Luc ) activity. C, effects of CRIF1 overexpression on t -BHQ-induced HO-1 luciferase reporter. Cells transfected with HO-1 reporter and FLAG-CRIF1 (except NRF2) for 16 h were incubated with either DMSO (control vehicle) or t -BHQ (100 μ m ) for 24 h before harvesting for measuring luciferase activity. D and E, effects of CRIF1 on NRF2 target gene expression. Total RNA was isolated from the MCF-7 cells infected with control Ad-GFP versus Ad-GFP-CRIF1 for 24 h and were treated with 100 μ m of t -BHQ ( D ) or 5 μ m of SFN ( E ). Real time PCR for HO-1 and GCLC was performed as described under “Experimental Procedures.”

    Techniques Used: Over Expression, Expressing, Luciferase, Plasmid Preparation, Binding Assay, Mutagenesis, Sequencing, Transfection, Activity Assay, Incubation, Isolation, Infection, Real-time Polymerase Chain Reaction

    CRIF1 regulates NRF2 protein levels via proteasome-mediated degradation. A, effect of CRIF1 overexpression on NRF2 protein levels. Cells (HEK293) were co-transfected for 24 h with three expression vectors (constant amounts of FLAG-NRF2 and pEGFP-N1 (the control vector) and increasing amounts of FLAG-CRIF1) and were analyzed on WB. B, effect of proteasomal inhibitors on CRIF1-driven down-regulation of NRF2 protein levels. Cells co-transfected as in A with the indicated expression vectors were incubated for an additional 4 h with proteasomal inhibitors (clasto-lactacystin β-lactone ( CLβL ) (10 μ m ), MG132 (10 μ m ), ALLN (50 μ m ), and epoxomicin (1 μ m )) and then analyzed by WB. Exogenous FLAG-CRIF1 and FLAG-NRF2 were identified by their different migration distances. C, effects of CRIF1 knockdown on NRF2 protein levels. Cells (MCF-7) pretreated with siRNA (control versus CRIF1–3 and -4) for 72 h were treated with SFN (5 μ m ) and harvested at the indicated times. Total cell lysates were used for WB analysis with anti-NRF2 and anti-CRIF1 mouse monoclonal antibodies. β-Actin was used as a loading and transfer control. D, nuclear extracts from cells in C were subjected to WB analysis. Lamin B1 was used as a control for nuclear fractionation and equal loading. The results of WB image quantification of triplicate experiments are shown as bar graphs on the bottom panels of C and D .
    Figure Legend Snippet: CRIF1 regulates NRF2 protein levels via proteasome-mediated degradation. A, effect of CRIF1 overexpression on NRF2 protein levels. Cells (HEK293) were co-transfected for 24 h with three expression vectors (constant amounts of FLAG-NRF2 and pEGFP-N1 (the control vector) and increasing amounts of FLAG-CRIF1) and were analyzed on WB. B, effect of proteasomal inhibitors on CRIF1-driven down-regulation of NRF2 protein levels. Cells co-transfected as in A with the indicated expression vectors were incubated for an additional 4 h with proteasomal inhibitors (clasto-lactacystin β-lactone ( CLβL ) (10 μ m ), MG132 (10 μ m ), ALLN (50 μ m ), and epoxomicin (1 μ m )) and then analyzed by WB. Exogenous FLAG-CRIF1 and FLAG-NRF2 were identified by their different migration distances. C, effects of CRIF1 knockdown on NRF2 protein levels. Cells (MCF-7) pretreated with siRNA (control versus CRIF1–3 and -4) for 72 h were treated with SFN (5 μ m ) and harvested at the indicated times. Total cell lysates were used for WB analysis with anti-NRF2 and anti-CRIF1 mouse monoclonal antibodies. β-Actin was used as a loading and transfer control. D, nuclear extracts from cells in C were subjected to WB analysis. Lamin B1 was used as a control for nuclear fractionation and equal loading. The results of WB image quantification of triplicate experiments are shown as bar graphs on the bottom panels of C and D .

    Techniques Used: Over Expression, Transfection, Expressing, Plasmid Preparation, Western Blot, Incubation, Migration, Fractionation

    17) Product Images from "Degeneration of Retinal ON Bipolar Cells Induced by Serum Including Autoantibody against TRPM1 in Mouse Model of Paraneoplastic Retinopathy"

    Article Title: Degeneration of Retinal ON Bipolar Cells Induced by Serum Including Autoantibody against TRPM1 in Mouse Model of Paraneoplastic Retinopathy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0081507

    Immunostaining with anti-human IgG antibody of mouse retina obtain 5 hours after intravitreal injection of serum. Immunostaining of mouse retina that was injected with control serum (A), with the patient's serum (B), and immunostaining of TRPM1 knockout mouse retina with the patient's serum (C). Human IgG was stained green. The regions indicated by the white boxes are enlarged below and stained with DAPI in blue (bottom of A, B and C). Fluorescein staining is prominent in the OPL (B). The scale bar is 25 μm in panels (A) to (C). (D) Immunoblots of transfected cell lysates using an antibody against Flag tag, antibody against mouse TRPM1, serum from PR patient, and serum from control subject. Arrowheads indicate the TRPM1-Flag protein bands and arrow indicates mouse TRPM1 protein. β-actin (β-act) was used for loading control. Patient serum had autoantibodies against both mouse and human TRPM1. Abbreviations: ILM - inner limiting membrane, RGC - retinal ganglion cells, IPL-inner plexiform layer, INL - inner nuclear layer, OPL - outer plexiform layer, ONL - outer nuclear layer.
    Figure Legend Snippet: Immunostaining with anti-human IgG antibody of mouse retina obtain 5 hours after intravitreal injection of serum. Immunostaining of mouse retina that was injected with control serum (A), with the patient's serum (B), and immunostaining of TRPM1 knockout mouse retina with the patient's serum (C). Human IgG was stained green. The regions indicated by the white boxes are enlarged below and stained with DAPI in blue (bottom of A, B and C). Fluorescein staining is prominent in the OPL (B). The scale bar is 25 μm in panels (A) to (C). (D) Immunoblots of transfected cell lysates using an antibody against Flag tag, antibody against mouse TRPM1, serum from PR patient, and serum from control subject. Arrowheads indicate the TRPM1-Flag protein bands and arrow indicates mouse TRPM1 protein. β-actin (β-act) was used for loading control. Patient serum had autoantibodies against both mouse and human TRPM1. Abbreviations: ILM - inner limiting membrane, RGC - retinal ganglion cells, IPL-inner plexiform layer, INL - inner nuclear layer, OPL - outer plexiform layer, ONL - outer nuclear layer.

    Techniques Used: Immunostaining, Injection, Knock-Out, Staining, Western Blot, Transfection, FLAG-tag, Activated Clotting Time Assay

    Distribution of ON bipolar cell marker (PKCα) in the retina after serum injection. Wild type mouse retina 24 hours after control serum injection (A) and wild type mouse retina 5 hours (B) and 24 hours (C) after intravitreal injection with the patient’s serum. Retina from TRPM1 knockout mouse 24 hours after intravitreal injection with the patient’s serum.(D) Mouse retinas were stained with anti PKCα antibody (green) and co-stained with DAPI (blue) in the high magnification micrographs (A-C, right). Photomicrographs of the regions outlined by the white boxes are enlarged either to the right or the below the original images. The peripheral retina is oriented to the left and the central retina to the right. PKCα staining can be seen in the entire retina at both 5 hours after the patient serum injection and 24 hours after control serum injection (A and B, arrows). But the PKCα staining is mainly absent 24 hours after the injection of the patient’s serum (C, asterisk) and remained in only the peripheral retina (C, arrows). PKCα staining can be seen 24 hours after the injection of the patient’s serum in TRPM1 knockout mouse retina (D arrows). High magnification micrograph showed that the PKC α positive cell bodies were located mainly on the photoreceptor side of the INL. The scale bars are: 60 µm for A left, B left, C upper left and D left; 20 µm for A, B, C and D right; 30 μm for C lower left.
    Figure Legend Snippet: Distribution of ON bipolar cell marker (PKCα) in the retina after serum injection. Wild type mouse retina 24 hours after control serum injection (A) and wild type mouse retina 5 hours (B) and 24 hours (C) after intravitreal injection with the patient’s serum. Retina from TRPM1 knockout mouse 24 hours after intravitreal injection with the patient’s serum.(D) Mouse retinas were stained with anti PKCα antibody (green) and co-stained with DAPI (blue) in the high magnification micrographs (A-C, right). Photomicrographs of the regions outlined by the white boxes are enlarged either to the right or the below the original images. The peripheral retina is oriented to the left and the central retina to the right. PKCα staining can be seen in the entire retina at both 5 hours after the patient serum injection and 24 hours after control serum injection (A and B, arrows). But the PKCα staining is mainly absent 24 hours after the injection of the patient’s serum (C, asterisk) and remained in only the peripheral retina (C, arrows). PKCα staining can be seen 24 hours after the injection of the patient’s serum in TRPM1 knockout mouse retina (D arrows). High magnification micrograph showed that the PKC α positive cell bodies were located mainly on the photoreceptor side of the INL. The scale bars are: 60 µm for A left, B left, C upper left and D left; 20 µm for A, B, C and D right; 30 μm for C lower left.

    Techniques Used: Marker, Injection, Knock-Out, Staining

    Light microscope (LM) and transmission electron microscope (TEM) photomicrographs of retinal sections. (A- C) LM photomicrographs of toluidine blue stained section obtained 5 hours after an intravitreal serum injection. (D - I) TEM photomicrographs of retinas at the same time point. (J - L) TUNEL (green) with Hoechst staining of retina obtained 1 day after the serum injection. Photomicrographs of retina from wild mouse that had the control serum injection (A, D, G, and J), the PR patient’s serum injection (B, E, H, and K) and retina from TRPM1 knockout mouse that had the patient’s serum injection. (C, F, I, and L). Photomicrographs of the regions outlined by the white boxes (D, E, and F) are enlarged below in (G, H, and I) respectively. Wild mouse retina after the injection of the patient’s serum shows many densely stained nuclei in the INL (B, arrow), and high magnification of TEM shows nuclear fragmentation and chromatin condensation of these cells (H, asterisk). Many TUNEL positive cells can be seen in the INL (K). The scale bar in the left row applies to the other two rows. The scale bar; C = 100 μm, F = 10 μm, I= 2 μm, and L = 10 μm.
    Figure Legend Snippet: Light microscope (LM) and transmission electron microscope (TEM) photomicrographs of retinal sections. (A- C) LM photomicrographs of toluidine blue stained section obtained 5 hours after an intravitreal serum injection. (D - I) TEM photomicrographs of retinas at the same time point. (J - L) TUNEL (green) with Hoechst staining of retina obtained 1 day after the serum injection. Photomicrographs of retina from wild mouse that had the control serum injection (A, D, G, and J), the PR patient’s serum injection (B, E, H, and K) and retina from TRPM1 knockout mouse that had the patient’s serum injection. (C, F, I, and L). Photomicrographs of the regions outlined by the white boxes (D, E, and F) are enlarged below in (G, H, and I) respectively. Wild mouse retina after the injection of the patient’s serum shows many densely stained nuclei in the INL (B, arrow), and high magnification of TEM shows nuclear fragmentation and chromatin condensation of these cells (H, asterisk). Many TUNEL positive cells can be seen in the INL (K). The scale bar in the left row applies to the other two rows. The scale bar; C = 100 μm, F = 10 μm, I= 2 μm, and L = 10 μm.

    Techniques Used: Light Microscopy, Transmission Assay, Microscopy, Transmission Electron Microscopy, Staining, Injection, TUNEL Assay, Knock-Out

    18) Product Images from "Receptor for Activated Protein Kinase C: Requirement for Efficient MicroRNA Function and Reduced Expression in Hepatocellular Carcinoma"

    Article Title: Receptor for Activated Protein Kinase C: Requirement for Efficient MicroRNA Function and Reduced Expression in Hepatocellular Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0024359

    RACK1 is required for miRNA function. A, Overexpression of miR122, miR140, and miR185 precursors suppressed activity of the corresponding reporters. RACK1 knockdown partially blocked these effects. miRNA reporter plasmids were transfected, with or without the corresponding miRNA precursor- and two types of siRACK1-expressing plasmids (siRACK1 (1) and (2)), into Huh7 cells. Values were normalized to those obtained from cells transfected with a miRNA precursor-non-expressing control vector, which were set to 1. Data represent the mean ± SD of three independent experiments. siGFP was used as a control, and it had no effect. Similar results were obtained using PLC/PRF/5 cells. B, CatA-Luc plasmids, which contain endogenous miR122 target sites derived from the CAT1 gene in its 3′-UTR, were transfected, with or without miR122 precursor- and siRACK1-expressing plasmids, into Huh7 cells. Data were generated and are presented as described in (A). Similar results were obtained using PLC/PRF/5 cells. C, Confirmation of the efficient knockdown of RACK1 expression in stable RACK1-knockdown Huh7 cells. 293T cell lysates were used as a positive control. D, miRNA function is impaired in stable RACK1-knockdown cells. miRNA185 reporter plasmids were transfected, with or without miR185 precursor-expressing plasmids, into control and RACK1-knockdown cells. Data were generated and are presented as described in (A). E, Endogenous miRNA function is impaired in RACK1-knockdown cells. miRNA185 reporter plasmids and CatA-Luc plasmids were transfected without miRNA precursor-expressing plasmids into control and RACK1-knockdown cells to assess endogenous miRNA function. Values were normalized to those obtained from control cells, which were set to 1. Data represent the mean ± SD of three independent experiments. *, p
    Figure Legend Snippet: RACK1 is required for miRNA function. A, Overexpression of miR122, miR140, and miR185 precursors suppressed activity of the corresponding reporters. RACK1 knockdown partially blocked these effects. miRNA reporter plasmids were transfected, with or without the corresponding miRNA precursor- and two types of siRACK1-expressing plasmids (siRACK1 (1) and (2)), into Huh7 cells. Values were normalized to those obtained from cells transfected with a miRNA precursor-non-expressing control vector, which were set to 1. Data represent the mean ± SD of three independent experiments. siGFP was used as a control, and it had no effect. Similar results were obtained using PLC/PRF/5 cells. B, CatA-Luc plasmids, which contain endogenous miR122 target sites derived from the CAT1 gene in its 3′-UTR, were transfected, with or without miR122 precursor- and siRACK1-expressing plasmids, into Huh7 cells. Data were generated and are presented as described in (A). Similar results were obtained using PLC/PRF/5 cells. C, Confirmation of the efficient knockdown of RACK1 expression in stable RACK1-knockdown Huh7 cells. 293T cell lysates were used as a positive control. D, miRNA function is impaired in stable RACK1-knockdown cells. miRNA185 reporter plasmids were transfected, with or without miR185 precursor-expressing plasmids, into control and RACK1-knockdown cells. Data were generated and are presented as described in (A). E, Endogenous miRNA function is impaired in RACK1-knockdown cells. miRNA185 reporter plasmids and CatA-Luc plasmids were transfected without miRNA precursor-expressing plasmids into control and RACK1-knockdown cells to assess endogenous miRNA function. Values were normalized to those obtained from control cells, which were set to 1. Data represent the mean ± SD of three independent experiments. *, p

    Techniques Used: Over Expression, Activity Assay, Transfection, Expressing, Plasmid Preparation, Planar Chromatography, Derivative Assay, Generated, Positive Control

    RACK1 binds to KSRP and may be involved in the recruitment of mature miRNAs to the RISC. A, Huh7 cells were transiently transfected with a control vector or a myc-tagged RACK1-expressing plasmid with or without a flag-tagged Dicer-expressing plasmid. Myc-tagged RACK1 was immunoprecipitated using anti-myc agarose. Normal mouse IgG was used as a control for immunoprecipitation. Co-precipitated proteins were blotted using antibodies against the indicated proteins. Five percent of the total cell lysates was loaded as “input.” Representative results from two independent experiments are shown. Similar results were obtained using 293T cells. B, Endogenous RACK1 in Huh7 cells was immunoprecipitated using anti-RACK1 antibody and Protein A/G Sepharose. Normal mouse IgG was used as a control for immunoprecipitation. Coprecipitated proteins were blotted using antibodies against the indicated proteins. Five percent of the total cell lysate was loaded as “input.” Representative results from two independent experiments are shown. C, Levels of mature miR122 and miR185 in Ago2-containing complexes were reduced in RACK1-knockdown cells. Mature miRNA levels were measured in RNA samples isolated from Ago2-containing complexes from control Huh7 (NC) cells and RACK1-knockdown (siRACK1) cells. miRNA levels were calculated as relative ratios. Data represent the mean ± SD of six independent experiments. *, p
    Figure Legend Snippet: RACK1 binds to KSRP and may be involved in the recruitment of mature miRNAs to the RISC. A, Huh7 cells were transiently transfected with a control vector or a myc-tagged RACK1-expressing plasmid with or without a flag-tagged Dicer-expressing plasmid. Myc-tagged RACK1 was immunoprecipitated using anti-myc agarose. Normal mouse IgG was used as a control for immunoprecipitation. Co-precipitated proteins were blotted using antibodies against the indicated proteins. Five percent of the total cell lysates was loaded as “input.” Representative results from two independent experiments are shown. Similar results were obtained using 293T cells. B, Endogenous RACK1 in Huh7 cells was immunoprecipitated using anti-RACK1 antibody and Protein A/G Sepharose. Normal mouse IgG was used as a control for immunoprecipitation. Coprecipitated proteins were blotted using antibodies against the indicated proteins. Five percent of the total cell lysate was loaded as “input.” Representative results from two independent experiments are shown. C, Levels of mature miR122 and miR185 in Ago2-containing complexes were reduced in RACK1-knockdown cells. Mature miRNA levels were measured in RNA samples isolated from Ago2-containing complexes from control Huh7 (NC) cells and RACK1-knockdown (siRACK1) cells. miRNA levels were calculated as relative ratios. Data represent the mean ± SD of six independent experiments. *, p

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

    RACK1 functions after miRNA maturation and before the silencing machinery. A, miRNA maturation was not impaired in RACK1-knockdown cells. Total RNA was isolated from control and RACK1-knockdown cells. Levels of mature miR122 were measured and normalized to the level of U6 snRNA. Relative ratios were calculated by adjusting the value for each miRNA in control cells to 1. Data represent the mean ± SD of six independent experiments. B, Artificial synthetic miRNA oligonucleotides function appropriately in RACK1-knockdown cells. Control Huh7 cells and RACK1-knockdown cells were transfected with miR122 reporter plasmids with or without synthetic corresponding miR122 oligonucleotides and non-corresponding miR185 oligonucleotides (to verify specificity). Values were normalized to those obtained from the cells transfected with control synthetic oligonucleotides, which were set to 1. Data represent the mean ± SD of three independent experiments. *, p
    Figure Legend Snippet: RACK1 functions after miRNA maturation and before the silencing machinery. A, miRNA maturation was not impaired in RACK1-knockdown cells. Total RNA was isolated from control and RACK1-knockdown cells. Levels of mature miR122 were measured and normalized to the level of U6 snRNA. Relative ratios were calculated by adjusting the value for each miRNA in control cells to 1. Data represent the mean ± SD of six independent experiments. B, Artificial synthetic miRNA oligonucleotides function appropriately in RACK1-knockdown cells. Control Huh7 cells and RACK1-knockdown cells were transfected with miR122 reporter plasmids with or without synthetic corresponding miR122 oligonucleotides and non-corresponding miR185 oligonucleotides (to verify specificity). Values were normalized to those obtained from the cells transfected with control synthetic oligonucleotides, which were set to 1. Data represent the mean ± SD of three independent experiments. *, p

    Techniques Used: Isolation, Transfection

    RACK1 expression is reduced in HCC. A, Immunohistochemical analysis of RACK1 protein expression in HCC and non-cancerous surrounding tissues. While strong staining was observed in the cytoplasm of hepatocytes in non-cancerous liver tissues (upper left (one panel)), HCC cells were stained more weakly (upper right). Lower panels: magnified images of the highlighted regions in the corresponding upper panels. Three representative cases are shown. Scale bar, 500 µm. B, Comparable expression of RACK1 in colon carcinoma tissues (right images) and non-cancerous surrounding tissues (left). Two representative cases are shown. Scale bar, 500 µm. C, Comparison of RACK1 staining in cancers and healthy surrounding tissues. Eight types of cancers were examined. Percentages of cases in which RACK1 expression was lower in cancerous tissues than in healthy tissues were calculated. The number of cases of each type of cancer studied is indicated.
    Figure Legend Snippet: RACK1 expression is reduced in HCC. A, Immunohistochemical analysis of RACK1 protein expression in HCC and non-cancerous surrounding tissues. While strong staining was observed in the cytoplasm of hepatocytes in non-cancerous liver tissues (upper left (one panel)), HCC cells were stained more weakly (upper right). Lower panels: magnified images of the highlighted regions in the corresponding upper panels. Three representative cases are shown. Scale bar, 500 µm. B, Comparable expression of RACK1 in colon carcinoma tissues (right images) and non-cancerous surrounding tissues (left). Two representative cases are shown. Scale bar, 500 µm. C, Comparison of RACK1 staining in cancers and healthy surrounding tissues. Eight types of cancers were examined. Percentages of cases in which RACK1 expression was lower in cancerous tissues than in healthy tissues were calculated. The number of cases of each type of cancer studied is indicated.

    Techniques Used: Expressing, Immunohistochemistry, Staining

    19) Product Images from "Bim and Bmf Synergize To Induce Apoptosis in Neisseria Gonorrhoeae Infection"

    Article Title: Bim and Bmf Synergize To Induce Apoptosis in Neisseria Gonorrhoeae Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000348

    Model of Bim-dependent and Bmf-dependent apoptosis during Ngo-induced apoptosis. Ngo infection leads to a Rac-dependent activation of JNK-1 and a concurrent alteration of the cytoskeletal morphology. Upon JNK-1–mediated phosphorylation, the cytoskeleton-attached proapoptotic proteins Bim and Bmf are released. Subsequently, the antiapoptotic effects of Mcl-1 and Bcl-X L are abrogated by Bim and Bmf, respectively, leading to the activation of Bak and Bax and cell death.
    Figure Legend Snippet: Model of Bim-dependent and Bmf-dependent apoptosis during Ngo-induced apoptosis. Ngo infection leads to a Rac-dependent activation of JNK-1 and a concurrent alteration of the cytoskeletal morphology. Upon JNK-1–mediated phosphorylation, the cytoskeleton-attached proapoptotic proteins Bim and Bmf are released. Subsequently, the antiapoptotic effects of Mcl-1 and Bcl-X L are abrogated by Bim and Bmf, respectively, leading to the activation of Bak and Bax and cell death.

    Techniques Used: Infection, Activation Assay

    Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-X L . (A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).
    Figure Legend Snippet: Bim-specific and Bmf-specific targeting of Mcl-1 and Bcl-X L . (A,B) The activation of Bak and Bax upon siRNA-mediated knockdown was visualized 15 h post-infection by immunoprecipitation with conformation-specific antibodies followed by SDS-PAGE and immunodetection with the indicated antibodies. (C) The network of Bcl-2 family proteins was analyzed by caspase activation assays after single or double knockdowns, 15 h post-infection. Shown are the means±SD of three independent experiments. (D,E) Knockdowns were validated by Western blot using the indicated antibodies (D) and qRT-PCR (E).

    Techniques Used: Activation Assay, Infection, Immunoprecipitation, SDS Page, Immunodetection, Western Blot, Quantitative RT-PCR

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

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

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.438762

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

    Techniques Used:

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

    Techniques Used:

    21) Product Images from "Maintenance of Endothelial Guanosine Triphosphate Cyclohydrolase I Ameliorates Diabetic Nephropathy"

    Article Title: Maintenance of Endothelial Guanosine Triphosphate Cyclohydrolase I Ameliorates Diabetic Nephropathy

    Journal: Journal of the American Society of Nephrology : JASN

    doi: 10.1681/ASN.2012080783

    Histologic findings and immunostaining of α-SMA and podocin. (A) Glomerular morphologic changes (upper panel, periodic acid–Schiff staining; lower panel, collagen IV staining). (B) Evaluation of glomerular size. (C) Evaluation of glomerular
    Figure Legend Snippet: Histologic findings and immunostaining of α-SMA and podocin. (A) Glomerular morphologic changes (upper panel, periodic acid–Schiff staining; lower panel, collagen IV staining). (B) Evaluation of glomerular size. (C) Evaluation of glomerular

    Techniques Used: Immunostaining, Staining

    22) Product Images from "Hirsutella sinensis mycelium attenuates bleomycin-induced pulmonary inflammation and fibrosis in vivo"

    Article Title: Hirsutella sinensis mycelium attenuates bleomycin-induced pulmonary inflammation and fibrosis in vivo

    Journal: Scientific Reports

    doi: 10.1038/srep15282

    HSM inhibits TGF-β1, collagen 3α1, and α-SMA expression in lung tissues of BLM–treated mice. ( a,c ) mRNA levels of collagen 3α1 and TGF-β1 in lung tissues from each group of mice on day 21 were determined by quantitative real-time RT-PCR. All data are presented as fold changes of gene expression normalized to β-actin. ( b,d ) Western blotting detection of collagen 3α1 and TGF-β1 expression from lung tissues of each group of mice on day 21. Increased collagen 3α1 and TGF-β1 levels were observed in BLM–treated mice (BLM + EtOH) compared with control mice (PBS + EtOH). ( e ) Representative α-SMA staining of lung tissue sections from control and experimental groups at day 21. α-SMA-positive cells (myofibroblasts and airway smooth muscle cells) decreased in HSM-pretreated mice compared with BLM-treated mice (scale bars = 50 μm). ( f ) Western blotting detection of α-SMA expression from lung tissues of each group of mice on day 21. The blots were analyzed by densitometry and normalized to β-actin. Data are presented as means ± SEM of at least three separate experiments. # P
    Figure Legend Snippet: HSM inhibits TGF-β1, collagen 3α1, and α-SMA expression in lung tissues of BLM–treated mice. ( a,c ) mRNA levels of collagen 3α1 and TGF-β1 in lung tissues from each group of mice on day 21 were determined by quantitative real-time RT-PCR. All data are presented as fold changes of gene expression normalized to β-actin. ( b,d ) Western blotting detection of collagen 3α1 and TGF-β1 expression from lung tissues of each group of mice on day 21. Increased collagen 3α1 and TGF-β1 levels were observed in BLM–treated mice (BLM + EtOH) compared with control mice (PBS + EtOH). ( e ) Representative α-SMA staining of lung tissue sections from control and experimental groups at day 21. α-SMA-positive cells (myofibroblasts and airway smooth muscle cells) decreased in HSM-pretreated mice compared with BLM-treated mice (scale bars = 50 μm). ( f ) Western blotting detection of α-SMA expression from lung tissues of each group of mice on day 21. The blots were analyzed by densitometry and normalized to β-actin. Data are presented as means ± SEM of at least three separate experiments. # P

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

    Anti-fibrotic effects of HSM ethanol extract on the production of myofibroblast marker proteins α-SMA and fibronectin through suppression of Smad2/3 and Akt signaling pathways in MRC-5 human lung fibroblasts. Cells were treated with various concentrations (1, 2, and 5%) of HSM extract for 24 h ( a ) or 48 h ( b ), and cell viability was measured using the MTT assay. ( c,d ) Cells were serum-starved overnight, before treatment with varying concentrations (1 and 2%) of HSM extract for 24 h and incubation with TGF-β1 (5 ng/ml) for 24 h. Expression of the myofibroblast markers α-SMA and fibronectin were evaluated in whole cell lysates by Western blotting. β-actin was used as a loading control. Relative protein levels were quantified by scanning densitometry and were normalized to β-actin. ( e,f ) Cells were pretreated with HSM extract (1 and 2%), followed by treatment with TGF-β1 (5 ng/ml) for another 6 h. Phosphorylated and total Smad2/3 and Akt were measured by Western blotting against phospho-Smad2/3, Smad2/3, phospho-Akt and Akt. Blots were analyzed by densitometry and the results were expressed as relative units. Data shown represent means ± SEM of three experiments performed in duplicate. # P
    Figure Legend Snippet: Anti-fibrotic effects of HSM ethanol extract on the production of myofibroblast marker proteins α-SMA and fibronectin through suppression of Smad2/3 and Akt signaling pathways in MRC-5 human lung fibroblasts. Cells were treated with various concentrations (1, 2, and 5%) of HSM extract for 24 h ( a ) or 48 h ( b ), and cell viability was measured using the MTT assay. ( c,d ) Cells were serum-starved overnight, before treatment with varying concentrations (1 and 2%) of HSM extract for 24 h and incubation with TGF-β1 (5 ng/ml) for 24 h. Expression of the myofibroblast markers α-SMA and fibronectin were evaluated in whole cell lysates by Western blotting. β-actin was used as a loading control. Relative protein levels were quantified by scanning densitometry and were normalized to β-actin. ( e,f ) Cells were pretreated with HSM extract (1 and 2%), followed by treatment with TGF-β1 (5 ng/ml) for another 6 h. Phosphorylated and total Smad2/3 and Akt were measured by Western blotting against phospho-Smad2/3, Smad2/3, phospho-Akt and Akt. Blots were analyzed by densitometry and the results were expressed as relative units. Data shown represent means ± SEM of three experiments performed in duplicate. # P

    Techniques Used: Marker, MTT Assay, Incubation, Expressing, Western Blot

    23) Product Images from "Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4"

    Article Title: Biochemical Properties of a Novel Cysteine Protease of Plasmodium vivax, Vivapain-4

    Journal: PLoS Neglected Tropical Diseases

    doi: 10.1371/journal.pntd.0000849

    Reactivity of rVX-4 against macromolecular substrates. ( A ) Processing of P. vivax plasmepsin 4 (PvPM4) by rVX-4. Recombinant PvPM 4 (20 µg) was incubated with rVX-4 (50 nM) supplemented with 10 mM DTT at different pH values with or without pepstatin A (10 µM) or E-64 (1 µM) for 3 h at 37°C. The reactants were analyzed by 12% SDS-PAGE. ( B ) Comparison of hemoglobinolytic activity of VX-2, VX-3 and VX-4. Native human hemoglobin was incubated with the respective enzymes in appropriate buffers (pH ranges 5.0–7.5) supplemented with 1 mM GSH for 3 h at 37°C, after which resolved by 10% SDS-PAGE. ( C ) Hydrolysis of erythrocyte membrane proteins by rVX-4 at different pHs. Fresh erythrocyte ghosts were incubated with rVX-4 in appropriate buffers (pHs 5.0–7.5) for 3 h at 37°C and reaction products were analyzed by 10% SDS-PAGE. Molecular masses in kDa are shown to the right. ( D ) Western blotting of erythrocyte ghost proteins. The reactions were done at pH 7 and 7.5. The reactants were separated by 10% SDS-PAGE, transferred to a PVDF membrane and probed with specific antibodies against human erythrocyte spectrin (1∶500), band 3 (1∶30000) and actin (1∶1000) followed by horseradish peroxidase conjugated anti-human IgG (1∶1000). The blots were developed with 4C1N. C, control without enzyme.
    Figure Legend Snippet: Reactivity of rVX-4 against macromolecular substrates. ( A ) Processing of P. vivax plasmepsin 4 (PvPM4) by rVX-4. Recombinant PvPM 4 (20 µg) was incubated with rVX-4 (50 nM) supplemented with 10 mM DTT at different pH values with or without pepstatin A (10 µM) or E-64 (1 µM) for 3 h at 37°C. The reactants were analyzed by 12% SDS-PAGE. ( B ) Comparison of hemoglobinolytic activity of VX-2, VX-3 and VX-4. Native human hemoglobin was incubated with the respective enzymes in appropriate buffers (pH ranges 5.0–7.5) supplemented with 1 mM GSH for 3 h at 37°C, after which resolved by 10% SDS-PAGE. ( C ) Hydrolysis of erythrocyte membrane proteins by rVX-4 at different pHs. Fresh erythrocyte ghosts were incubated with rVX-4 in appropriate buffers (pHs 5.0–7.5) for 3 h at 37°C and reaction products were analyzed by 10% SDS-PAGE. Molecular masses in kDa are shown to the right. ( D ) Western blotting of erythrocyte ghost proteins. The reactions were done at pH 7 and 7.5. The reactants were separated by 10% SDS-PAGE, transferred to a PVDF membrane and probed with specific antibodies against human erythrocyte spectrin (1∶500), band 3 (1∶30000) and actin (1∶1000) followed by horseradish peroxidase conjugated anti-human IgG (1∶1000). The blots were developed with 4C1N. C, control without enzyme.

    Techniques Used: Recombinant, Incubation, SDS Page, Activity Assay, Western Blot

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

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

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.4029-03.2004

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

    Techniques Used: Expressing, Mouse Assay

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

    Techniques Used: Expressing, Western Blot

    25) Product Images from "A novel anti-proliferative role of HMGA2 in induction of apoptosis through caspase 2 in primary human fibroblast cells"

    Article Title: A novel anti-proliferative role of HMGA2 in induction of apoptosis through caspase 2 in primary human fibroblast cells

    Journal: Bioscience Reports

    doi: 10.1042/BSR20140112

    Caspase-2 was crucial for HMGA2-induced apoptosis in WI38 cells ( A ) DNA-damage foci labelled by γH2A in WI38 cells expressing HMGA2 at day 5. The percentage of γH2A positive cells was shown in brackets. Scale bar: 20 μm. ( B ) Western blots showing the increase of γH2A and activated caspase 2 in WI38 cells expressing HMGA2 at indicated time. ( C ) Caspase-2-deficient WI38 cells partially escaped from HMGA2-induced cell death as manifested in relative survival percentages calculated from MTT assay at day 8. ( D ) Attenuated levels of cleaved caspase 2 and -3 in cells treated as in ( C ). ( E ) Immunofluorescence images showing that the cytochrome c release from mitochondria induced by HMGA2 was interrupted by shCaspase 2. The percentage of cytochrome c diffusion positive cells was shown in brackets. Scale bar: 50 μm.
    Figure Legend Snippet: Caspase-2 was crucial for HMGA2-induced apoptosis in WI38 cells ( A ) DNA-damage foci labelled by γH2A in WI38 cells expressing HMGA2 at day 5. The percentage of γH2A positive cells was shown in brackets. Scale bar: 20 μm. ( B ) Western blots showing the increase of γH2A and activated caspase 2 in WI38 cells expressing HMGA2 at indicated time. ( C ) Caspase-2-deficient WI38 cells partially escaped from HMGA2-induced cell death as manifested in relative survival percentages calculated from MTT assay at day 8. ( D ) Attenuated levels of cleaved caspase 2 and -3 in cells treated as in ( C ). ( E ) Immunofluorescence images showing that the cytochrome c release from mitochondria induced by HMGA2 was interrupted by shCaspase 2. The percentage of cytochrome c diffusion positive cells was shown in brackets. Scale bar: 50 μm.

    Techniques Used: Expressing, Western Blot, MTT Assay, Immunofluorescence, Diffusion-based Assay

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0033287

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

    Techniques Used: Migration, Transfection, Isolation

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

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

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

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

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

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

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

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

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

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

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-8-56

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

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

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

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

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004393

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

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

    29) Product Images from "CCN family protein 2 (CCN2) promotes the early differentiation, but inhibits the terminal differentiation of skeletal myoblasts"

    Article Title: CCN family protein 2 (CCN2) promotes the early differentiation, but inhibits the terminal differentiation of skeletal myoblasts

    Journal: Journal of Biochemistry

    doi: 10.1093/jb/mvu056

    Differentiation of myoblasts from Ccn2 -deficient mice is impaired. ( A ) Primary myoblasts isolated from gastrocnemius muscle of WT and Ccn2 -deficient (KO) mice at E18.5 were treated with rCCN2 at the concentration indicated for 7 days. Western blot analysis was performed by using anti-MyoD and anti-β-actin antibodies. ( B ) WT and KO myoblasts were induced to differentiate for 7 days in differentiation medium. Then, western blot analysis was performed by using anti-integrin α5 and anti-β-actin antibodies. ( C ) WT and KO myoblasts were induced to differentiate for 7 days. The cells were thereafter fixed, and immunofluorescence analysis by using anti-desmin antibody was performed. Bar represents 100 µm. ( D ) WT and KO myoblasts were induced to differentiate for 6 days, and real-time RT–PCR analysis was performed. The amounts of MyoD , MHC , and Myostatin mRNAs were normalized to that of Gapdh mRNA and the bars represent means and standard deviations of four sets of independent cultures. Exact P -values are determined by Student’s t test.
    Figure Legend Snippet: Differentiation of myoblasts from Ccn2 -deficient mice is impaired. ( A ) Primary myoblasts isolated from gastrocnemius muscle of WT and Ccn2 -deficient (KO) mice at E18.5 were treated with rCCN2 at the concentration indicated for 7 days. Western blot analysis was performed by using anti-MyoD and anti-β-actin antibodies. ( B ) WT and KO myoblasts were induced to differentiate for 7 days in differentiation medium. Then, western blot analysis was performed by using anti-integrin α5 and anti-β-actin antibodies. ( C ) WT and KO myoblasts were induced to differentiate for 7 days. The cells were thereafter fixed, and immunofluorescence analysis by using anti-desmin antibody was performed. Bar represents 100 µm. ( D ) WT and KO myoblasts were induced to differentiate for 6 days, and real-time RT–PCR analysis was performed. The amounts of MyoD , MHC , and Myostatin mRNAs were normalized to that of Gapdh mRNA and the bars represent means and standard deviations of four sets of independent cultures. Exact P -values are determined by Student’s t test.

    Techniques Used: Mouse Assay, Isolation, Concentration Assay, Western Blot, Immunofluorescence, Quantitative RT-PCR

    CCN2 production is increased in C2C12 cells treated with TNFα. ( A ) C2C12 cells were stimulated by 20 ng/ml TNFα in differentiation medium. At the indicated time the cell lysates were collected and western blot analysis was performed by using anti-CCN2 and β-actin antibodies. ( B ) C2C12 cells were treated with TNFα at the indicated concentrations for 2 days. Then, western blot analysis was similarly performed. ( C ) Quantification of the results obtained from the experiments for panel A at 48 h in triplicates. * P
    Figure Legend Snippet: CCN2 production is increased in C2C12 cells treated with TNFα. ( A ) C2C12 cells were stimulated by 20 ng/ml TNFα in differentiation medium. At the indicated time the cell lysates were collected and western blot analysis was performed by using anti-CCN2 and β-actin antibodies. ( B ) C2C12 cells were treated with TNFα at the indicated concentrations for 2 days. Then, western blot analysis was similarly performed. ( C ) Quantification of the results obtained from the experiments for panel A at 48 h in triplicates. * P

    Techniques Used: Western Blot

    CCN2 regulates the gene expression of Desmin (A), MHC (B), and Myostatin (C) in C2C12 cells. C2C12 cells were stimulated with rCCN2 at the concentrations indicated for 4 days, and real-time PCR analysis was performed. The amounts of these gene products were normalized to that of Gapdh with respect to the control sample (ratio = 1.0). Bars represent means and standard deviations of results obtained from eight to nine different cultures for each sample. * P
    Figure Legend Snippet: CCN2 regulates the gene expression of Desmin (A), MHC (B), and Myostatin (C) in C2C12 cells. C2C12 cells were stimulated with rCCN2 at the concentrations indicated for 4 days, and real-time PCR analysis was performed. The amounts of these gene products were normalized to that of Gapdh with respect to the control sample (ratio = 1.0). Bars represent means and standard deviations of results obtained from eight to nine different cultures for each sample. * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    CCN2 regulates the gene expression and protein production of MyoD and myogenin in C2C12 cells. ( A ) C2C12 cells were stimulated with rCCN2 at the indicated concentrations in differentiation medium. After 1 and 2 days, the gene expression and protein production of MyoD were examined, respectively. * P
    Figure Legend Snippet: CCN2 regulates the gene expression and protein production of MyoD and myogenin in C2C12 cells. ( A ) C2C12 cells were stimulated with rCCN2 at the indicated concentrations in differentiation medium. After 1 and 2 days, the gene expression and protein production of MyoD were examined, respectively. * P

    Techniques Used: Expressing

    CCN2 promotes the cell proliferation of C2C12 cells. ( A ) C2C12 cells were stimulated for 16 h with rCCN2 at the indicated concentrations in differentiation medium. Then, cell proliferation assay was performed. Each bar shows the mean value and standard deviation of the results from nine wells. ** P
    Figure Legend Snippet: CCN2 promotes the cell proliferation of C2C12 cells. ( A ) C2C12 cells were stimulated for 16 h with rCCN2 at the indicated concentrations in differentiation medium. Then, cell proliferation assay was performed. Each bar shows the mean value and standard deviation of the results from nine wells. ** P

    Techniques Used: Proliferation Assay, Standard Deviation

    Both proliferation and differentiation of Ccn2 -deficient (KO) myoblasts is impaired in vivo . ( A ) Double immunoreactivity for PCNA and MyoD was detected in the sections of the skeletal muscle from E18.5 WT and KO littermates by immunofluorescence analysis, and the bottom graph shows the means and standard deviations of the number of double positive cells from three different views. ( B ) Immunoreactivity for desmin was detected in the sections from WT and KO skeletal muscle tissues, and the bottom graph shows the means and standard deviations of areas of muscle fibers stained with anti-desmin antibody. * P
    Figure Legend Snippet: Both proliferation and differentiation of Ccn2 -deficient (KO) myoblasts is impaired in vivo . ( A ) Double immunoreactivity for PCNA and MyoD was detected in the sections of the skeletal muscle from E18.5 WT and KO littermates by immunofluorescence analysis, and the bottom graph shows the means and standard deviations of the number of double positive cells from three different views. ( B ) Immunoreactivity for desmin was detected in the sections from WT and KO skeletal muscle tissues, and the bottom graph shows the means and standard deviations of areas of muscle fibers stained with anti-desmin antibody. * P

    Techniques Used: In Vivo, Immunofluorescence, Staining

    30) Product Images from "MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing"

    Article Title: MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

    Journal: The American Journal of Pathology

    doi: 10.2353/ajpath.2009.081080

    Analysis of CTGF degradation by immunoblotting. A: Homogenate supernatants were obtained from wound samples in wild-type (WT) and MMP-13 KO mice on days 3, 5, and 10 and subjected to immunoblotting. Intact CTGF and its degradation fragments are indicated. β-actin is used as a loading control. B: Ratio of intact CTGF to total CTGF. The ratio was determined by densitometrical analysis of the bands of intact and its degradation fragments ( n = 3). Bars = mean ± SE. ** P
    Figure Legend Snippet: Analysis of CTGF degradation by immunoblotting. A: Homogenate supernatants were obtained from wound samples in wild-type (WT) and MMP-13 KO mice on days 3, 5, and 10 and subjected to immunoblotting. Intact CTGF and its degradation fragments are indicated. β-actin is used as a loading control. B: Ratio of intact CTGF to total CTGF. The ratio was determined by densitometrical analysis of the bands of intact and its degradation fragments ( n = 3). Bars = mean ± SE. ** P

    Techniques Used: Mouse Assay

    31) Product Images from "Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells"

    Article Title: Gene array analysis of neural crest cells identifies transcription factors necessary for direct conversion of embryonic fibroblasts into neural crest cells

    Journal: Biology Open

    doi: 10.1242/bio.015735

    Converted SOX10+ NC cells differentiated into neural cells, glial cells, osteocytes and adipocytes. (A) Experimental scheme for the differentiation of SOX10+ NC cells converted from MEFs. Converted SOX10+ NC cells were cultured under NC cell culture conditions with hypoxia. After expansion for six days, the converted SOX10+ NC cells were cultured in each type of differentiation medium. (B-F) Immunostaining image of the differentiated SOX10+ NC cells. TuJ1+ neural cells, GFAP+ glial cells, and α smooth muscle actin (αSMA)+ smooth muscle cells were differentiated from isolated SOX10+ NC cells in the NC cell differentiation medium (B). In specific differentiation medium, the converted SOX10+ NC cells differentiated into TuJ1+, peripherin+, and nestin+ neural cells (C); GFAP+ and S100β+ glial cells (D); osteocytes with alkaline phosphatase activity (ALP) and positive staining with Alizarin Red (E); and adipocytes with positive Oil Red O staining (F). Scale bar=50 µm in B left, C,D,E left, F; 100 µm in B right, E right. Nuclei were stained with Hoechst 33258 (Blue). (G) RT-PCR analysis of neural cells, glial cells, osteocytes and adipocytes differentiated from Sox10+ NC cells. N, neural cells differentiated from Sox10+ NC cells; G, glial cells differentiated from Sox10+ NC cells; Os, osteocytes differentiated from Sox10+ NC cells; Ad, adipocytes differentiated from Sox10+ NC cells.
    Figure Legend Snippet: Converted SOX10+ NC cells differentiated into neural cells, glial cells, osteocytes and adipocytes. (A) Experimental scheme for the differentiation of SOX10+ NC cells converted from MEFs. Converted SOX10+ NC cells were cultured under NC cell culture conditions with hypoxia. After expansion for six days, the converted SOX10+ NC cells were cultured in each type of differentiation medium. (B-F) Immunostaining image of the differentiated SOX10+ NC cells. TuJ1+ neural cells, GFAP+ glial cells, and α smooth muscle actin (αSMA)+ smooth muscle cells were differentiated from isolated SOX10+ NC cells in the NC cell differentiation medium (B). In specific differentiation medium, the converted SOX10+ NC cells differentiated into TuJ1+, peripherin+, and nestin+ neural cells (C); GFAP+ and S100β+ glial cells (D); osteocytes with alkaline phosphatase activity (ALP) and positive staining with Alizarin Red (E); and adipocytes with positive Oil Red O staining (F). Scale bar=50 µm in B left, C,D,E left, F; 100 µm in B right, E right. Nuclei were stained with Hoechst 33258 (Blue). (G) RT-PCR analysis of neural cells, glial cells, osteocytes and adipocytes differentiated from Sox10+ NC cells. N, neural cells differentiated from Sox10+ NC cells; G, glial cells differentiated from Sox10+ NC cells; Os, osteocytes differentiated from Sox10+ NC cells; Ad, adipocytes differentiated from Sox10+ NC cells.

    Techniques Used: Cell Culture, Immunostaining, Isolation, Cell Differentiation, Activity Assay, ALP Assay, Staining, Reverse Transcription Polymerase Chain Reaction

    32) Product Images from "Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation"

    Article Title: Caveolin-1 Is Required for Kinase Suppressor of Ras 1 (KSR1)-Mediated Extracellular Signal-Regulated Kinase 1/2 Activation, H-RasV12-Induced Senescence, and Transformation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01633-13

    KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.
    Figure Legend Snippet: KSR1 interacts with caveolin-1. (A) KSR1 +/+ or KSR1 −/− MEFs expressing H-Ras V12 or control vectors were either lysed (WCL) or fractionated into cytoplasmic (Cyto), membrane Triton-soluble (MTS), or membrane Triton-insoluble (MTI) fractions (see Materials and Methods). Lysates were then probed with the indicated antibodies to assess whether KSR1 was required to drive MEK1/2 and ERK1/2 into the caveolin-1 signaling compartment. (B) Schematic diagram of murine KSR1 showing a putative caveolar binding motif (CBM) in the kinase-like domain and the regions that mediate Raf, MEK, and ERK interaction. (C) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated (IP) for caveolin-1, and the immunoprecipitates were probed for KSR1 to assess the KSR1–caveolin-1 interaction. IB, immunoblotting. (D) KSR1–caveolin-1 interaction examined using a proximity ligation assay (PLA) in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1 or CBM. KSR1 −/− MEFs expressing GFP were used as a negative control. PCI, phase contrast image. (E) Quantification of cells demonstrating congregation of bright spots along the periphery in serum-starved and EGF-stimulated KSR1 −/− MEFs expressing KSR1. (F) Panels I and II show images of PLA-generated fluorescence in KSR1 −/− MEFs expressing WT KSR1 before and after EGF stimulation. Panels III and IV show higher magnifications of the boxed regions in panels I and II, respectively.

    Techniques Used: Expressing, Binding Assay, Plasmid Preparation, Immunoprecipitation, Proximity Ligation Assay, Negative Control, Generated, Fluorescence

    The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P
    Figure Legend Snippet: The KSR1–caveolin-1 interaction promotes EGF-stimulated ERK1/2 activation. (A) WCLs from immortalized KSR1 −/− MEFs expressing control vector, KSR1, or CBM were immunoprecipitated for caveolin-1, and the immunoprecipitates were probed for MEK1/2 and ERK1/2 to assess the KSR1-MEK1/2 and KSR1-ERK1/2 interaction. (B) WCLs from 293T cells transfected with vector, FLAG-tagged KSR1, or FLAG-tagged CBM were immunoprecipitated with anti-FLAG antibodies and subjected to Western blotting for B-Raf and c-Raf to assess KSR1–B-Raf and KSR1–c-Raf interactions. The arrow denotes the band specific for c-Raf. The asterisk indicates s a nonspecific band. (C) Triplicate wells of immortalized KSR1 −/− MEFs expressing either KSR1 or KSR1.CBM were treated with 100 ng/ml EGF for the indicated times. ERK1/2 phosphorylation levels were determined in situ for ERK1 and phospho-ERK1/2 with a Li-Cor Odyssey system. Data are expressed as the ratio of phospho-ERK1/2 to ERK1. Data are expressed as means ± standard deviations from three independent experiments. ****, P

    Techniques Used: Activation Assay, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection, Western Blot, In Situ

    33) Product Images from "Efavirenz Promotes ?-Secretase Expression and Increased A?1-40,42 via Oxidative Stress and Reduced Microglial Phagocytosis: Implications for HIV Associated Neurocognitive Disorders (HAND)"

    Article Title: Efavirenz Promotes ?-Secretase Expression and Increased A?1-40,42 via Oxidative Stress and Reduced Microglial Phagocytosis: Implications for HIV Associated Neurocognitive Disorders (HAND)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0095500

    EFV/3TC/AZT increases soluble Aβ levels in Tg2576 mice via BACE-1 activation in vivo. ( A ) Aβ 40, 42 peptides were analyzed in brain homogenates from 8 month old Tg2576 mice by ELISA (n = 5 mice for each group). One-way ANOVA followed by post hoc comparison revealed significant differences between control (Tg2576mice treated with PBS) and EFV or EFV/3TC/AZT -treated Tg2576 mice ( P
    Figure Legend Snippet: EFV/3TC/AZT increases soluble Aβ levels in Tg2576 mice via BACE-1 activation in vivo. ( A ) Aβ 40, 42 peptides were analyzed in brain homogenates from 8 month old Tg2576 mice by ELISA (n = 5 mice for each group). One-way ANOVA followed by post hoc comparison revealed significant differences between control (Tg2576mice treated with PBS) and EFV or EFV/3TC/AZT -treated Tg2576 mice ( P

    Techniques Used: Mouse Assay, Activation Assay, In Vivo, Enzyme-linked Immunosorbent Assay

    Proposed mechanism of EFV neurotoxicity. Our present work suggests that EFV promotes an increase in Aβ in vitro and in vivo on both the production and clearance fronts via its inhibition of neurnoal MMP resulting in reduced ATP stores and thus a high ROS environment in the CNS. Previous studies indicate such high ROS microenvironments in the CNS promote BACE-1 APP processing and also inhibit microglial Aβ clearance functions. These events in turn all promote production of Aβ species. (*Note: Red arrows = inhibition, Green arrows = promotion).
    Figure Legend Snippet: Proposed mechanism of EFV neurotoxicity. Our present work suggests that EFV promotes an increase in Aβ in vitro and in vivo on both the production and clearance fronts via its inhibition of neurnoal MMP resulting in reduced ATP stores and thus a high ROS environment in the CNS. Previous studies indicate such high ROS microenvironments in the CNS promote BACE-1 APP processing and also inhibit microglial Aβ clearance functions. These events in turn all promote production of Aβ species. (*Note: Red arrows = inhibition, Green arrows = promotion).

    Techniques Used: In Vitro, In Vivo, Inhibition

    EFV or EFV/3TC/AZT treatment promotes Aβ generation in cultured neuronal cells via BACE-1 activation in vitro. Aβ species were analyzed in cell lysates from SweAPP N2a cells ( A ) by ELISA. Data are represented as the mean ± of a percentage of Aβ peptides secreted 24 h after 3TC, AZT, EFV, or 3TC/EFV/AZT administration, relative fold over control (PBS treated). Significant increases in Aβ were observed in EFV or EFV/3TC/AZT treated cells were observed compared to control (*** P
    Figure Legend Snippet: EFV or EFV/3TC/AZT treatment promotes Aβ generation in cultured neuronal cells via BACE-1 activation in vitro. Aβ species were analyzed in cell lysates from SweAPP N2a cells ( A ) by ELISA. Data are represented as the mean ± of a percentage of Aβ peptides secreted 24 h after 3TC, AZT, EFV, or 3TC/EFV/AZT administration, relative fold over control (PBS treated). Significant increases in Aβ were observed in EFV or EFV/3TC/AZT treated cells were observed compared to control (*** P

    Techniques Used: Cell Culture, Activation Assay, In Vitro, Enzyme-linked Immunosorbent Assay

    34) Product Images from "Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation"

    Article Title: Role of STIM1- and Orai1-mediated Ca2+ entry in Ca2+-induced epidermal keratinocyte differentiation

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.115980

    Both STIM1 and Orai1 are important for maintenance of intracellular stored Ca 2+ in HaCaT cells cultured in low Ca 2+ medium. Average time courses of Ca 2+ release from whole intracellular Ca 2+ stores by ionomycin in siRNA-transfected HaCaT cells cultured in low ( A ) or high ( B ) Ca 2+ medium. ( C ) Peak [Ca 2+ ] rises after whole intracellular store depletion by 20 µM ionomycin. Data are means ± s.e.m. from three independent experiments. *** P
    Figure Legend Snippet: Both STIM1 and Orai1 are important for maintenance of intracellular stored Ca 2+ in HaCaT cells cultured in low Ca 2+ medium. Average time courses of Ca 2+ release from whole intracellular Ca 2+ stores by ionomycin in siRNA-transfected HaCaT cells cultured in low ( A ) or high ( B ) Ca 2+ medium. ( C ) Peak [Ca 2+ ] rises after whole intracellular store depletion by 20 µM ionomycin. Data are means ± s.e.m. from three independent experiments. *** P

    Techniques Used: Cell Culture, Transfection

    STIM1 and Orai1 protein expression during keratinocyte differentiation. ( A ) Western blotting showing STIM1 and Orai1 protein expression in siRNA-transfected HaCaT cells before and after Ca 2+ ). The full lane westerns for both STIM1 and Orai1 are shown in supplementary material Fig. S1. ( B , C ) Densitometric analysis of STIM1 (B) or Orai1 (C) expression. Intensities of bands of STIM1 or Orai1 were normalized by those of actin. Relative expression to the sample at time 0 from siControl-transfected cells are shown. Data are means ± s.e.m. from three independent experiments.
    Figure Legend Snippet: STIM1 and Orai1 protein expression during keratinocyte differentiation. ( A ) Western blotting showing STIM1 and Orai1 protein expression in siRNA-transfected HaCaT cells before and after Ca 2+ ). The full lane westerns for both STIM1 and Orai1 are shown in supplementary material Fig. S1. ( B , C ) Densitometric analysis of STIM1 (B) or Orai1 (C) expression. Intensities of bands of STIM1 or Orai1 were normalized by those of actin. Relative expression to the sample at time 0 from siControl-transfected cells are shown. Data are means ± s.e.m. from three independent experiments.

    Techniques Used: Expressing, Western Blot, Transfection

    Both STIM1 and Orai1 are indispensable for TG-induced SOCE in HaCaT cells. ( A , B ) Average time courses of TG-induced Ca 2+ responses in siRNA-transfected HaCaT cells cultured in KGM-2 containing 0.03 mM Ca 2+ (A) or 1.8 mM Ca 2+ (B). Right panels showed peak [Ca 2+ ] rises attributable to Ca 2+ release and influx evoked by TG. Data are means ± s.e.m. from three independent experiments. ** P
    Figure Legend Snippet: Both STIM1 and Orai1 are indispensable for TG-induced SOCE in HaCaT cells. ( A , B ) Average time courses of TG-induced Ca 2+ responses in siRNA-transfected HaCaT cells cultured in KGM-2 containing 0.03 mM Ca 2+ (A) or 1.8 mM Ca 2+ (B). Right panels showed peak [Ca 2+ ] rises attributable to Ca 2+ release and influx evoked by TG. Data are means ± s.e.m. from three independent experiments. ** P

    Techniques Used: Transfection, Cell Culture

    Both STIM1 and Orai1 are required for Ca 2+ -switch-induced differentiation marker expression in HaCaT cells. Ca 2+ -switch-induced expression of an early differentiation marker gene KRT1 in siRNA-transfected HaCaT cells. Fold increases of KRT1 expression relative to the sample at time 0 from siControl-transfected cell are shown. Data are means ± s.e.m. from four independent experiments. *** P
    Figure Legend Snippet: Both STIM1 and Orai1 are required for Ca 2+ -switch-induced differentiation marker expression in HaCaT cells. Ca 2+ -switch-induced expression of an early differentiation marker gene KRT1 in siRNA-transfected HaCaT cells. Fold increases of KRT1 expression relative to the sample at time 0 from siControl-transfected cell are shown. Data are means ± s.e.m. from four independent experiments. *** P

    Techniques Used: Marker, Expressing, Transfection

    Both STIM1 and Orai1 are required for Ca 2+ -switch-induced Ca 2+ responses in HaCaT cells. Average time courses of 10 mM extracellular Ca 2+ -induced Ca 2+ responses in siRNA-transfected HaCaT cells (left). Peak [Ca 2+ ] rises of Ca 2+ -switch-induced Ca 2+ responses (right). Data are means ± s.e.m. from three independent experiments. ** P
    Figure Legend Snippet: Both STIM1 and Orai1 are required for Ca 2+ -switch-induced Ca 2+ responses in HaCaT cells. Average time courses of 10 mM extracellular Ca 2+ -induced Ca 2+ responses in siRNA-transfected HaCaT cells (left). Peak [Ca 2+ ] rises of Ca 2+ -switch-induced Ca 2+ responses (right). Data are means ± s.e.m. from three independent experiments. ** P

    Techniques Used: Transfection

    STIM1 and Orai1 are important for normal cell growth and Ca 2+ -switch-induced growth arrest. Cells were cultured for 72 hours in 0.03 mM or 1.8 mM Ca 2+ -containing KGM-2. Cell growth was analyzed by the XTT cell proliferation assay. XTT activity in siSTIM1- and siOrai1-transfected cells is shown relative to that in siControl-transfected HaCaT cells in control low Ca 2+ medium for 72 hours. Data are means ± s.e.m. from three independent experiments. *** P
    Figure Legend Snippet: STIM1 and Orai1 are important for normal cell growth and Ca 2+ -switch-induced growth arrest. Cells were cultured for 72 hours in 0.03 mM or 1.8 mM Ca 2+ -containing KGM-2. Cell growth was analyzed by the XTT cell proliferation assay. XTT activity in siSTIM1- and siOrai1-transfected cells is shown relative to that in siControl-transfected HaCaT cells in control low Ca 2+ medium for 72 hours. Data are means ± s.e.m. from three independent experiments. *** P

    Techniques Used: Cell Culture, Proliferation Assay, Activity Assay, Transfection

    Both STIM1 and Orai1 are important for I CRAC in HaCaT. ( A ) Average time courses of I CRAC induced by intracellular dialysis of 20 µM Ins(1,4,5) P 3 and 10 mM BAPTA. Switches of extracellular solution are indicated in the horizontal bar above the traces. ( B ) Peak inward current recorded at the holding of −120 mV. Data are means ± s.e.m.; siControl, n = 4; siSTIM1 and siOrai1, n = 3. *** P
    Figure Legend Snippet: Both STIM1 and Orai1 are important for I CRAC in HaCaT. ( A ) Average time courses of I CRAC induced by intracellular dialysis of 20 µM Ins(1,4,5) P 3 and 10 mM BAPTA. Switches of extracellular solution are indicated in the horizontal bar above the traces. ( B ) Peak inward current recorded at the holding of −120 mV. Data are means ± s.e.m.; siControl, n = 4; siSTIM1 and siOrai1, n = 3. *** P

    Techniques Used:

    35) Product Images from "Upregulation of actin cytoskeleton via myocardin leads to increased expression of type 1 collagen"

    Article Title: Upregulation of actin cytoskeleton via myocardin leads to increased expression of type 1 collagen

    Journal: Laboratory investigation; a journal of technical methods and pathology

    doi: 10.1038/labinvest.2017.96

    Overexpression of myocardin promotes HSC myofibroblast differentiation and liver fibrosis. (A) A schematic map for overexpression of myocardin with 4 doses of CCl 4 . 5 days after the final dose of CCl 4, HSCs were isolated and cultured overnight; representative phase contrast images are shown (arrow indicates an activated HSC). (B, C) Total RNA was extracted from the same cells and RT-PCR was performed to detect SM α-actin and Col.1α1 mRNA (n=3, p
    Figure Legend Snippet: Overexpression of myocardin promotes HSC myofibroblast differentiation and liver fibrosis. (A) A schematic map for overexpression of myocardin with 4 doses of CCl 4 . 5 days after the final dose of CCl 4, HSCs were isolated and cultured overnight; representative phase contrast images are shown (arrow indicates an activated HSC). (B, C) Total RNA was extracted from the same cells and RT-PCR was performed to detect SM α-actin and Col.1α1 mRNA (n=3, p

    Techniques Used: Over Expression, Isolation, Cell Culture, Reverse Transcription Polymerase Chain Reaction

    Myocardin-induced differential regulation of actin isoforms is CArG box and ATTA, CCAAT box dependent. (A) CArG box sequences from rat SM α-actin, cytoplasmic-γ-actin (Cyto-γ-actin) and β-actin promoters are aligned. Site mutations are shown in bold letters (CC in CArG boxes were replaced by TT). (B) Activated HSCs were cotransfected with luciferase reporter constructs containing wild type or the CArG box mutation as in (A) and a myocardin expression plasmid (Myocd) or empty vector. Cells were harvested 2 days later to detect for promoter activity. (C) After growth for 2 days after isolation, HSCs were exposed to the indicated adenoviral vectors for 3 days and 2 days later, they were subjected to EMSA to measure the effect of myocardin on SRF binding activity. SRF binding and supershifted bands are highlighted by arrows (lane 1, 2, 5, 8: nuclear extracts from HSCs infected with Ad-control virus; lane 3, 6, 9: nuclear extracts from HSCs infected with Ad-myocd virus; lane 4, 7, 10: nuclear extracts from HSCs infected with Ad-myocd-DN virus). Representative data from 3 independent experiments are shown. (D) EMSA was performed using nuclear extracts (10 μg) from activated HSCs and the same amount of different actin CArG box probes (1 × 10 5 cpm) as indicated. SRF binding and supershifted complexes were indicated by arrows (upper panel). Nuclear extracts were probed by anti-SRF antibody as loading control (bottom panel). Representative data from 3 independent experiments are shown. (E) A schematic diagram of wild type and mutant actin isoform promoters is shown (mutated nucleotides were indicated below the consensus sequences); (F) luciferase assays were performed as in (B). (G) A schematic diagram of exchanged elements (ATTA and CCAAT boxes) among SM α-actin, cytoplasmic-γ-actin (cyto-γ-actin) and β-actin promoters is shown; (H) luciferase activity assay was performed as in (B). (n=3, * p
    Figure Legend Snippet: Myocardin-induced differential regulation of actin isoforms is CArG box and ATTA, CCAAT box dependent. (A) CArG box sequences from rat SM α-actin, cytoplasmic-γ-actin (Cyto-γ-actin) and β-actin promoters are aligned. Site mutations are shown in bold letters (CC in CArG boxes were replaced by TT). (B) Activated HSCs were cotransfected with luciferase reporter constructs containing wild type or the CArG box mutation as in (A) and a myocardin expression plasmid (Myocd) or empty vector. Cells were harvested 2 days later to detect for promoter activity. (C) After growth for 2 days after isolation, HSCs were exposed to the indicated adenoviral vectors for 3 days and 2 days later, they were subjected to EMSA to measure the effect of myocardin on SRF binding activity. SRF binding and supershifted bands are highlighted by arrows (lane 1, 2, 5, 8: nuclear extracts from HSCs infected with Ad-control virus; lane 3, 6, 9: nuclear extracts from HSCs infected with Ad-myocd virus; lane 4, 7, 10: nuclear extracts from HSCs infected with Ad-myocd-DN virus). Representative data from 3 independent experiments are shown. (D) EMSA was performed using nuclear extracts (10 μg) from activated HSCs and the same amount of different actin CArG box probes (1 × 10 5 cpm) as indicated. SRF binding and supershifted complexes were indicated by arrows (upper panel). Nuclear extracts were probed by anti-SRF antibody as loading control (bottom panel). Representative data from 3 independent experiments are shown. (E) A schematic diagram of wild type and mutant actin isoform promoters is shown (mutated nucleotides were indicated below the consensus sequences); (F) luciferase assays were performed as in (B). (G) A schematic diagram of exchanged elements (ATTA and CCAAT boxes) among SM α-actin, cytoplasmic-γ-actin (cyto-γ-actin) and β-actin promoters is shown; (H) luciferase activity assay was performed as in (B). (n=3, * p

    Techniques Used: Luciferase, Construct, Mutagenesis, Expressing, Plasmid Preparation, Activity Assay, Isolation, Binding Assay, Infection

    36) Product Images from "Acute phase protein, α – 1- acid glycoprotein (AGP-1), has differential effects on TLR-2 and TLR-4 mediated responses"

    Article Title: Acute phase protein, α – 1- acid glycoprotein (AGP-1), has differential effects on TLR-2 and TLR-4 mediated responses

    Journal: bioRxiv

    doi: 10.1101/634295

    Schematic representation of selective inhibition of MAPKs by AGP-1 induced by LPS but not BLP AGP-1 selectively inhibits p-38, JNK and ERK MAPKs induced by TLR-4 activation thereby delaying the mortality. However, AGP-1 had no significant effect on the TLR-2 induced MAPK activation but potentiated the BLP mediated mortality.
    Figure Legend Snippet: Schematic representation of selective inhibition of MAPKs by AGP-1 induced by LPS but not BLP AGP-1 selectively inhibits p-38, JNK and ERK MAPKs induced by TLR-4 activation thereby delaying the mortality. However, AGP-1 had no significant effect on the TLR-2 induced MAPK activation but potentiated the BLP mediated mortality.

    Techniques Used: Inhibition, Activation Assay

    AGP-1 selectively modulates TLR-2 mediated mortality Swiss albino mice were divided into an indicated number of groups containing six animals each and the mice were injected intraperitoneally with the stated concentrations of BLP and D-GalN in the presence/absence of AGP-1 in a total volume of 500 μl. The survival time was monitored for 24 hours. (A) The graph represents the percentage of survival with respect to time of the groups of mice receiving indicated amounts of BLP and LPS (inset) (15 mg/kg) with or without AGP-1. (B) Time of death of individual animals receiving BLP with or without AGP-1. Inset: LPS (15 mg/kg) was used as a positive control. The result is a representative of three independent experiments. ***p
    Figure Legend Snippet: AGP-1 selectively modulates TLR-2 mediated mortality Swiss albino mice were divided into an indicated number of groups containing six animals each and the mice were injected intraperitoneally with the stated concentrations of BLP and D-GalN in the presence/absence of AGP-1 in a total volume of 500 μl. The survival time was monitored for 24 hours. (A) The graph represents the percentage of survival with respect to time of the groups of mice receiving indicated amounts of BLP and LPS (inset) (15 mg/kg) with or without AGP-1. (B) Time of death of individual animals receiving BLP with or without AGP-1. Inset: LPS (15 mg/kg) was used as a positive control. The result is a representative of three independent experiments. ***p

    Techniques Used: Mouse Assay, Injection, Positive Control

    AGP-1 inhibits TLR-4 mediated but potentiates TLR-2 mediated adhesion of neutrophils (A) Neutrophils loaded with calcein-AM in HBSS/A was treated respectively with vehicle, 0.5% serum, LPS/BLP (100□ng/ml) in the presence/absence of AGP-1 (25 – 100 μg/ml). The mixture was incubated for 60□min at 37□°C and the non-adherent PMNs were removed by washing with HBSS. The adherent PMNs were visualized under a fluorescent microscope at a magnification of 40X. Although AGP-1 alone activated neutrophils dose-dependently, it inhibited TLR-4 mediated but potentiated TLR-2 mediated β2-integrin-mediated adhesion of neutrophils to the gelatinous substrate. (B) PMN activation was quantified by counting the cells per field using ImageJ software as explained under “Methods”. ***p
    Figure Legend Snippet: AGP-1 inhibits TLR-4 mediated but potentiates TLR-2 mediated adhesion of neutrophils (A) Neutrophils loaded with calcein-AM in HBSS/A was treated respectively with vehicle, 0.5% serum, LPS/BLP (100□ng/ml) in the presence/absence of AGP-1 (25 – 100 μg/ml). The mixture was incubated for 60□min at 37□°C and the non-adherent PMNs were removed by washing with HBSS. The adherent PMNs were visualized under a fluorescent microscope at a magnification of 40X. Although AGP-1 alone activated neutrophils dose-dependently, it inhibited TLR-4 mediated but potentiated TLR-2 mediated β2-integrin-mediated adhesion of neutrophils to the gelatinous substrate. (B) PMN activation was quantified by counting the cells per field using ImageJ software as explained under “Methods”. ***p

    Techniques Used: Incubation, Microscopy, Activation Assay, Software

    Isolation and characterization of AGP-1 from Human serum (A) Elution profile of human serum proteins on Cibacron blue column. Human serum (2 ml, 80 mg/ml) was loaded on to Cibacron blue pseudo-affinity column and the proteins eluted with 10 mM phosphate buffer pH 7.8 (B) Elution profile of concentrated Cibacron blue column void volume fraction from DEAE-cellulose eluted using 30 mM acetate buffer (pH 5.0) containing 0 – 2 M NaCl. The fraction containing AGP-1 is encircled in red. 7.5% SDS-PAGE analysis of AGP-1 after each step of purification was performed and the gel was subjected to both PAS staining (C) and Coomassie brilliant blue staining (D) and western blot analysis (E) using human AGP-1 monoclonal antibody. (F) AGP-1 isolated from human serum (hAGP1) and commercially available AGP-1 (cAGP1) was subjected to mass spectral analysis and compared to the AGP-1 protein sequence in the NCBI database. PSMs, AAs, and HT represent peptide spectral matches, amino acids, and hits, respectively.
    Figure Legend Snippet: Isolation and characterization of AGP-1 from Human serum (A) Elution profile of human serum proteins on Cibacron blue column. Human serum (2 ml, 80 mg/ml) was loaded on to Cibacron blue pseudo-affinity column and the proteins eluted with 10 mM phosphate buffer pH 7.8 (B) Elution profile of concentrated Cibacron blue column void volume fraction from DEAE-cellulose eluted using 30 mM acetate buffer (pH 5.0) containing 0 – 2 M NaCl. The fraction containing AGP-1 is encircled in red. 7.5% SDS-PAGE analysis of AGP-1 after each step of purification was performed and the gel was subjected to both PAS staining (C) and Coomassie brilliant blue staining (D) and western blot analysis (E) using human AGP-1 monoclonal antibody. (F) AGP-1 isolated from human serum (hAGP1) and commercially available AGP-1 (cAGP1) was subjected to mass spectral analysis and compared to the AGP-1 protein sequence in the NCBI database. PSMs, AAs, and HT represent peptide spectral matches, amino acids, and hits, respectively.

    Techniques Used: Isolation, Affinity Column, SDS Page, Purification, Staining, Western Blot, Sequencing, Atomic Absorption Spectroscopy

    Activation of the MAPK pathway in mouse peritoneal macrophages by AGP-1 and its effect on LPS and BLP induced MAPK pathway (A) AGP-1 affects phospho – p38, JNK and ERK MAP kinases. (B) AGP-1 inhibits LPS – induced MAPK pathway activation but not BLP – mediated MAPKs activation. Mouse peritoneal macrophage lysates from the stated amounts of LPS and BLP in the presence and absence of AGP-1 treated cells were prepared using RIPA buffer and immunoblots were developed using specific primary and appropriate secondary antibodies for phospho – forms of MAPKs (p38, JNK and ERK) and β-actin and visualized as described in ‘Methods’. (C) and (D) densitometric analysis of the (A) and (B) blots respectively. Densitometry was done using ImageJ software. The blots are representative of 3 different blots. The data shown are mean ±SEM ***p
    Figure Legend Snippet: Activation of the MAPK pathway in mouse peritoneal macrophages by AGP-1 and its effect on LPS and BLP induced MAPK pathway (A) AGP-1 affects phospho – p38, JNK and ERK MAP kinases. (B) AGP-1 inhibits LPS – induced MAPK pathway activation but not BLP – mediated MAPKs activation. Mouse peritoneal macrophage lysates from the stated amounts of LPS and BLP in the presence and absence of AGP-1 treated cells were prepared using RIPA buffer and immunoblots were developed using specific primary and appropriate secondary antibodies for phospho – forms of MAPKs (p38, JNK and ERK) and β-actin and visualized as described in ‘Methods’. (C) and (D) densitometric analysis of the (A) and (B) blots respectively. Densitometry was done using ImageJ software. The blots are representative of 3 different blots. The data shown are mean ±SEM ***p

    Techniques Used: Activation Assay, Western Blot, Software

    37) Product Images from "Heme-Sensing Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics"

    Article Title: Heme-Sensing Pathway Modulates Susceptibility of Poor Prognosis B-Lineage Acute Leukemia to BH3-Mimetics

    Journal: bioRxiv

    doi: 10.1101/2020.04.10.036319

    HRI is required for DHA induced MCL-1 repression and synergism with BH3-mimetics. ( A ) BCR-ABL + B-ALL cells were treated with DHA for 16h and protein expression was determined by immunoblotting with indicated antibodies. (B) Wild-type (WT) or Eif2ak1 -KO (lacking HRI) BCR-ABL + B-ALL cells were treated with DHA for 16h and protein expression was determined by immunoblotting with indicated antibodies. (C) Purified HRI protein was incubated with 20 µM heme, 20 µM DHA, or a combination of the two at room temperature for 30 min. Absorbance was then measured at 420 nm to detect the presence of a Soret peak which is indicative of protein binding. Data are the average of three experiments and error bars are SEM. Unpaired t-test indicates significance between HRI+Heme vs. HRI+Heme+DHA p
    Figure Legend Snippet: HRI is required for DHA induced MCL-1 repression and synergism with BH3-mimetics. ( A ) BCR-ABL + B-ALL cells were treated with DHA for 16h and protein expression was determined by immunoblotting with indicated antibodies. (B) Wild-type (WT) or Eif2ak1 -KO (lacking HRI) BCR-ABL + B-ALL cells were treated with DHA for 16h and protein expression was determined by immunoblotting with indicated antibodies. (C) Purified HRI protein was incubated with 20 µM heme, 20 µM DHA, or a combination of the two at room temperature for 30 min. Absorbance was then measured at 420 nm to detect the presence of a Soret peak which is indicative of protein binding. Data are the average of three experiments and error bars are SEM. Unpaired t-test indicates significance between HRI+Heme vs. HRI+Heme+DHA p

    Techniques Used: Expressing, Purification, Incubation, Protein Binding

    Direct activation of HRI represses MCL-1 and synergizes with BH3-mimetics. (A) Wild-type (WT) or Eif2ak1 -KO (HRI-deficient) BCR-ABL + B-ALL cells were treated with BTdCPU for 16h and protein expression was determined by immunoblotting with indicated antibodies. ( B and C ) WT or Eif2ak1 -KO BCR-ABL + B-ALL cells were treated with (B) ABT-263 or (C) ABT-199 (0, 40, 80, 160 nM) alone or in combination with the indicated concentrations of BTdCPU for 24h. Viable cells were measured using Annexin-V and propidium iodide staining. Data are the average of three experiments and error bars are SEM. Two-way ANOVA with Bonferroni multiple comparison indicates significance P
    Figure Legend Snippet: Direct activation of HRI represses MCL-1 and synergizes with BH3-mimetics. (A) Wild-type (WT) or Eif2ak1 -KO (HRI-deficient) BCR-ABL + B-ALL cells were treated with BTdCPU for 16h and protein expression was determined by immunoblotting with indicated antibodies. ( B and C ) WT or Eif2ak1 -KO BCR-ABL + B-ALL cells were treated with (B) ABT-263 or (C) ABT-199 (0, 40, 80, 160 nM) alone or in combination with the indicated concentrations of BTdCPU for 24h. Viable cells were measured using Annexin-V and propidium iodide staining. Data are the average of three experiments and error bars are SEM. Two-way ANOVA with Bonferroni multiple comparison indicates significance P

    Techniques Used: Activation Assay, Expressing, Staining

    Densitometry and synergy analysis of HRI activation ex vivo . (A) Densitometry of MCL-1 expression in bone marrow cells harvested from BTdCPU treated mice (related to Fig. 6C ). Data are the average of 3 separate animals and error bars are SEM. Unpaired t-test indicates significance between vehicle and BTdCPU p
    Figure Legend Snippet: Densitometry and synergy analysis of HRI activation ex vivo . (A) Densitometry of MCL-1 expression in bone marrow cells harvested from BTdCPU treated mice (related to Fig. 6C ). Data are the average of 3 separate animals and error bars are SEM. Unpaired t-test indicates significance between vehicle and BTdCPU p

    Techniques Used: Activation Assay, Ex Vivo, Expressing, Mouse Assay

    HRI activation represses MCL-1 and synergizes with BH3-mimetics in Ph + and Ph-like ALL cell lines. ( A ) Human Ph + BV-173 cells were treated with the indicated concentrations of DHA or BTdCPU for 24h and protein expression was determined by western blotting. ( B ) Wild-type (WT) or a knockdown pool of EIF2AK1 -KO (lacking HRI) BV-173 cells were treated with DMSO (Control, C), DHA (5 µM), BTdCPU (BTd, 5 µM), or Thapsigargin (Thp, 100 nM) for 24h and protein expression was determined by western blotting. (C) WT or EIF2AK1 -KO BV-173 cells were treated with ABT-263 (0, 5, 10, 20 nM) or ABT-199 (0, 2.5, 5, 10 nM) alone or in combination with the indicated concentrations of DHA or BTdCPU for 24h. Viable cells were measured using Annexin-V and propidium iodide staining. Data are the average of 3 experiments and error bars are SEM. Two-way ANOVA with Bonferroni multiple comparison indicates significance P
    Figure Legend Snippet: HRI activation represses MCL-1 and synergizes with BH3-mimetics in Ph + and Ph-like ALL cell lines. ( A ) Human Ph + BV-173 cells were treated with the indicated concentrations of DHA or BTdCPU for 24h and protein expression was determined by western blotting. ( B ) Wild-type (WT) or a knockdown pool of EIF2AK1 -KO (lacking HRI) BV-173 cells were treated with DMSO (Control, C), DHA (5 µM), BTdCPU (BTd, 5 µM), or Thapsigargin (Thp, 100 nM) for 24h and protein expression was determined by western blotting. (C) WT or EIF2AK1 -KO BV-173 cells were treated with ABT-263 (0, 5, 10, 20 nM) or ABT-199 (0, 2.5, 5, 10 nM) alone or in combination with the indicated concentrations of DHA or BTdCPU for 24h. Viable cells were measured using Annexin-V and propidium iodide staining. Data are the average of 3 experiments and error bars are SEM. Two-way ANOVA with Bonferroni multiple comparison indicates significance P

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

    38) Product Images from "D-Aspartate Upregulates DAAM1 Protein Levels in the Rat Testis and Induces Its Localization in Spermatogonia Nucleus"

    Article Title: D-Aspartate Upregulates DAAM1 Protein Levels in the Rat Testis and Induces Its Localization in Spermatogonia Nucleus

    Journal: Biomolecules

    doi: 10.3390/biom10050677

    DAAM1 protein levels and localization in cultured mouse GC-1 cells. ( A ) DAAM1 Western blot detection (123 kDa) in GC-1 cells at 30 min after D-Asp treatment and at time 0 (control, C); T = Testis. ( B ) DAAM1 protein levels are quantified using the ImageJ program and normalized with respect to β-actin (42 kDa). Values represent the means ± S.D. of three separate experiments. * p
    Figure Legend Snippet: DAAM1 protein levels and localization in cultured mouse GC-1 cells. ( A ) DAAM1 Western blot detection (123 kDa) in GC-1 cells at 30 min after D-Asp treatment and at time 0 (control, C); T = Testis. ( B ) DAAM1 protein levels are quantified using the ImageJ program and normalized with respect to β-actin (42 kDa). Values represent the means ± S.D. of three separate experiments. * p

    Techniques Used: Cell Culture, Western Blot

    DAAM1 (123 kDa) levels in cytosolic and nuclear protein fractions in D-Asp treated rats. ( A ) Western blot detection of DAAM1 protein in cytosolic and nuclear fractions of D-Asp-treated and control rat testis. To evaluate the purity of the samples, the nuclear protein levels of histone H3 (17 kDa) was detected. ( B ) The amount of DAAM1 was quantified using ImageJ program and normalized with respect to β-actin (42 kDa). Values represent the means ± S.D. of five samples. * p
    Figure Legend Snippet: DAAM1 (123 kDa) levels in cytosolic and nuclear protein fractions in D-Asp treated rats. ( A ) Western blot detection of DAAM1 protein in cytosolic and nuclear fractions of D-Asp-treated and control rat testis. To evaluate the purity of the samples, the nuclear protein levels of histone H3 (17 kDa) was detected. ( B ) The amount of DAAM1 was quantified using ImageJ program and normalized with respect to β-actin (42 kDa). Values represent the means ± S.D. of five samples. * p

    Techniques Used: Western Blot

    Schematic representation of domain architecture of rat DAAM1. ( A ) The main domains are represented: GTPase binding domain (GBD) domain (blue), Formin Homology 1 (FH1) domain (green) and Formin Homology 2 (FH2) domain (yellow). ( B ) Amino acid sequence of the predicted monopartite nuclear localization signal (NLS) (purple). ( C ) Amino acid sequence of the predicted bipartite NLS (red).
    Figure Legend Snippet: Schematic representation of domain architecture of rat DAAM1. ( A ) The main domains are represented: GTPase binding domain (GBD) domain (blue), Formin Homology 1 (FH1) domain (green) and Formin Homology 2 (FH2) domain (yellow). ( B ) Amino acid sequence of the predicted monopartite nuclear localization signal (NLS) (purple). ( C ) Amino acid sequence of the predicted bipartite NLS (red).

    Techniques Used: Binding Assay, Sequencing

    Testicular Disheveled-Associated-Activator of Morphogenesis1 (DAAM1) protein levels and localization in D-Asp treated rats. ( A ) Western blot analysis of DAAM1 (123 kDa) protein levels in the testis from D-Asp-treated and control rats. ( B ) The amount of DAAM1 was quantified using ImageJ program and normalized with respect to β-actin (42 kDa). Values represent the means ± S.D. of five samples. ** p
    Figure Legend Snippet: Testicular Disheveled-Associated-Activator of Morphogenesis1 (DAAM1) protein levels and localization in D-Asp treated rats. ( A ) Western blot analysis of DAAM1 (123 kDa) protein levels in the testis from D-Asp-treated and control rats. ( B ) The amount of DAAM1 was quantified using ImageJ program and normalized with respect to β-actin (42 kDa). Values represent the means ± S.D. of five samples. ** p

    Techniques Used: Western Blot

    39) Product Images from "Neuronal Per Arnt Sim (PAS) Domain Protein 4 (NPAS4) Regulates Neurite Outgrowth and Phosphorylation of Synapsin I *"

    Article Title: Neuronal Per Arnt Sim (PAS) Domain Protein 4 (NPAS4) Regulates Neurite Outgrowth and Phosphorylation of Synapsin I *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.413310

    Effect of DM treatment for 48 h on neurite outgrowth, NPAS4, and p-SYN I expression levels in Neuro2a cells. A, representative photographs showing control ( top ) and DM-treated cells ( bottom ). Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 261 for control and n = 251 for DM.). *, p
    Figure Legend Snippet: Effect of DM treatment for 48 h on neurite outgrowth, NPAS4, and p-SYN I expression levels in Neuro2a cells. A, representative photographs showing control ( top ) and DM-treated cells ( bottom ). Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 261 for control and n = 251 for DM.). *, p

    Techniques Used: Expressing

    NPAS4 overexpression increased NeuN, p-SYN I, and TUJ1 expression levels in Neuro2a cells. A, immunoblot analysis of NeuN expression levels induced by NPAS4 overexpression ( n = 10–11). B, immunoblot analysis of p-SYN I expression levels induced by NPAS4 overexpression ( n = 10–11). C, immunoblot analysis of TUJ1 expression levels induced by NPAS4 overexpression with DM ( n = 6). Values indicate the mean ± S.E. *, p
    Figure Legend Snippet: NPAS4 overexpression increased NeuN, p-SYN I, and TUJ1 expression levels in Neuro2a cells. A, immunoblot analysis of NeuN expression levels induced by NPAS4 overexpression ( n = 10–11). B, immunoblot analysis of p-SYN I expression levels induced by NPAS4 overexpression ( n = 10–11). C, immunoblot analysis of TUJ1 expression levels induced by NPAS4 overexpression with DM ( n = 6). Values indicate the mean ± S.E. *, p

    Techniques Used: Over Expression, Expressing

    PTZ-induced NPAS4, p-SYN I, SYN I, CDK5, and p35 expression in the prefrontal cortex of mice at different time points. A, immunoblot analysis of NPAS4, p-SYN I, SYN I, CDK5, and p35 expression levels in the prefrontal cortex of mice at various time points after PTZ injection. B, quantitative analysis of immunoblots ( A ). Values indicate the mean ± S.E. ( n = 4). *, p
    Figure Legend Snippet: PTZ-induced NPAS4, p-SYN I, SYN I, CDK5, and p35 expression in the prefrontal cortex of mice at different time points. A, immunoblot analysis of NPAS4, p-SYN I, SYN I, CDK5, and p35 expression levels in the prefrontal cortex of mice at various time points after PTZ injection. B, quantitative analysis of immunoblots ( A ). Values indicate the mean ± S.E. ( n = 4). *, p

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

    NPAS4 overexpression increased CDK5 and p-SYN I expression levels and the relationship between CDK5 and p-SYN I expression in Neuro2a cells. A, representative photographs showing immunocytochemical analysis of CDK5 and p-SYN I expression levels (72 h after transfection). Scale bar, 20 μm. B, quantitative analysis of CDK5 expression levels at 24 and 72 h after transfection ( left ). Values (the mean ± S.E.) indicate normalized CDK5 levels. ( n = 81 for Npas4 vector transfection (Npas4), n = 32 for mock vector transfection (Mock), and n = 74 for nontransfection (control), 24 h after transfection. n = 103 for Npas4 vector transfection (Npas4), n = 39 for mock vector transfection (Mock), and n = 154 for nontransfection (control), 72 h after transfection.) *, p
    Figure Legend Snippet: NPAS4 overexpression increased CDK5 and p-SYN I expression levels and the relationship between CDK5 and p-SYN I expression in Neuro2a cells. A, representative photographs showing immunocytochemical analysis of CDK5 and p-SYN I expression levels (72 h after transfection). Scale bar, 20 μm. B, quantitative analysis of CDK5 expression levels at 24 and 72 h after transfection ( left ). Values (the mean ± S.E.) indicate normalized CDK5 levels. ( n = 81 for Npas4 vector transfection (Npas4), n = 32 for mock vector transfection (Mock), and n = 74 for nontransfection (control), 24 h after transfection. n = 103 for Npas4 vector transfection (Npas4), n = 39 for mock vector transfection (Mock), and n = 154 for nontransfection (control), 72 h after transfection.) *, p

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation

    NPAS4 is a key factor in PTZ-induced phosphorylation of SYN I. A, immunoblot analysis of NPAS4, p-SYN I, and SYN I expression levels in the prefrontal cortex of mice 2 h after PTZ injection. B, quantitative analysis of immunoblots ( A ). Values indicate the mean ± S.E. ( n = 5–8). *, p
    Figure Legend Snippet: NPAS4 is a key factor in PTZ-induced phosphorylation of SYN I. A, immunoblot analysis of NPAS4, p-SYN I, and SYN I expression levels in the prefrontal cortex of mice 2 h after PTZ injection. B, quantitative analysis of immunoblots ( A ). Values indicate the mean ± S.E. ( n = 5–8). *, p

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

    Effect of NPAS4 knockdown on neurite outgrowth induced by DM in Neuro2a cells. A, representative photographs showing immunocytochemical analysis of siRNA- or scrRNA-transfected cells (GFP-positive). Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 79 for Npas4 siRNA transfection (siRNA), n = 81 for scrambled siRNA transfection (scrRNA), and n = 463 for nontransfection (control).). *, p
    Figure Legend Snippet: Effect of NPAS4 knockdown on neurite outgrowth induced by DM in Neuro2a cells. A, representative photographs showing immunocytochemical analysis of siRNA- or scrRNA-transfected cells (GFP-positive). Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 79 for Npas4 siRNA transfection (siRNA), n = 81 for scrambled siRNA transfection (scrRNA), and n = 463 for nontransfection (control).). *, p

    Techniques Used: Transfection

    Roscovitine inhibited neurite outgrowth in NPAS4-overexpressed Neuro2a cells. A, representative photographs showing immunocytochemical analysis of neurite outgrowth in Npas4 vector-transfected cells (Myc-positive) in the presence or absence of roscovitine treatment. Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 89 for Npas4 + DMSO; n = 120 for mock + DMSO; n = 545 for control + DMSO; n = 148 for Npas4 + roscovitine; n = 65 for mock + roscovitine, and n = 308 for control + roscovitine). *, p
    Figure Legend Snippet: Roscovitine inhibited neurite outgrowth in NPAS4-overexpressed Neuro2a cells. A, representative photographs showing immunocytochemical analysis of neurite outgrowth in Npas4 vector-transfected cells (Myc-positive) in the presence or absence of roscovitine treatment. Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 89 for Npas4 + DMSO; n = 120 for mock + DMSO; n = 545 for control + DMSO; n = 148 for Npas4 + roscovitine; n = 65 for mock + roscovitine, and n = 308 for control + roscovitine). *, p

    Techniques Used: Plasmid Preparation, Transfection

    NPAS4 overexpression increased p-SYN I expression levels in primary cultured hippocampal neurons. A, representative photographs showing immunocytochemical analysis of p-SYN I expression in Npas4 vector-transfected cells (Myc-positive). Scale bar, 20 μm. B, quantitative analysis of p-SYN I expression levels. Values (the mean ± S.E.) indicate normalized p-SYN I levels. ( n = 48 for Npas4 vector transfection (Npas4), n = 34 for mock vector transfection ( Mock ), and n = 77 for nontransfection ( Control )). *, p
    Figure Legend Snippet: NPAS4 overexpression increased p-SYN I expression levels in primary cultured hippocampal neurons. A, representative photographs showing immunocytochemical analysis of p-SYN I expression in Npas4 vector-transfected cells (Myc-positive). Scale bar, 20 μm. B, quantitative analysis of p-SYN I expression levels. Values (the mean ± S.E.) indicate normalized p-SYN I levels. ( n = 48 for Npas4 vector transfection (Npas4), n = 34 for mock vector transfection ( Mock ), and n = 77 for nontransfection ( Control )). *, p

    Techniques Used: Over Expression, Expressing, Cell Culture, Plasmid Preparation, Transfection

    NPAS4 overexpression increased CDK5 expression levels and kinase activity and roscovitine inhibited p-SYN I expression levels. A, immunoblot analysis of CDK5 expression levels induced by NPAS4 overexpression ( n = 3). Values indicate the mean ± S.E. *, p
    Figure Legend Snippet: NPAS4 overexpression increased CDK5 expression levels and kinase activity and roscovitine inhibited p-SYN I expression levels. A, immunoblot analysis of CDK5 expression levels induced by NPAS4 overexpression ( n = 3). Values indicate the mean ± S.E. *, p

    Techniques Used: Over Expression, Expressing, Activity Assay

    Effect of NPAS4 overexpression on neurite outgrowth induced by DM in Neuro2a cells. A, representative photographs showing immunocytochemical analysis of Npas4 - or mock vector-transfected cells (Myc-positive). Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 88 for Npas4 vector transfection (Npas4), n = 42 for mock vector transfection ( Mock ), and n = 160 for nontransfection ( Control ).) *, p
    Figure Legend Snippet: Effect of NPAS4 overexpression on neurite outgrowth induced by DM in Neuro2a cells. A, representative photographs showing immunocytochemical analysis of Npas4 - or mock vector-transfected cells (Myc-positive). Scale bar, 20 μm. B, quantitative analysis of neurite length. Values indicate the mean ± S.E. ( left ) and the cumulative frequency of neurite length ( right ). ( n = 88 for Npas4 vector transfection (Npas4), n = 42 for mock vector transfection ( Mock ), and n = 160 for nontransfection ( Control ).) *, p

    Techniques Used: Over Expression, Plasmid Preparation, Transfection

    40) Product Images from "(E)-2-Methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) Phenol Ameliorates LPS-Mediated Memory Impairment by Inhibition of STAT3 Pathway"

    Article Title: (E)-2-Methoxy-4-(3-(4-methoxyphenyl) prop-1-en-1-yl) Phenol Ameliorates LPS-Mediated Memory Impairment by Inhibition of STAT3 Pathway

    Journal: Neuromolecular Medicine

    doi: 10.1007/s12017-017-8469-3

    Inhibitory effect of MMPP on amyloidogenesis and STAT3 translocation in astrocytes and microglia cells. The expression of APP, BACE1 and C99 was detected by Western blotting using specific antibodies in astrocytes ( a ) and microglia cells ( b ). Each blot is representative of three experiments. The activity of β-secretase was investigated using assay kit in astrocytes ( c ) and microglia cells ( d ). Values are presented as mean ± S.D. of the three independent experiments performed in triplicate. # p
    Figure Legend Snippet: Inhibitory effect of MMPP on amyloidogenesis and STAT3 translocation in astrocytes and microglia cells. The expression of APP, BACE1 and C99 was detected by Western blotting using specific antibodies in astrocytes ( a ) and microglia cells ( b ). Each blot is representative of three experiments. The activity of β-secretase was investigated using assay kit in astrocytes ( c ) and microglia cells ( d ). Values are presented as mean ± S.D. of the three independent experiments performed in triplicate. # p

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

    Inhibitory effects of MMPP on accumulation of Aβ 1-42 in the brain of LPS-injected mice. ( a ) Aβ accumulation in the brains of LPS-injected mice was determined by thioflavin S staining. The levels of Aβ 1-42 in mice brain ( n = 5) were measured by ELISA ( b ). The activity of β-secretase in mice brain ( n = 5) was investigated using assay kit ( c ). The expression of APP, BACE1 and C99 was detected by Western blotting using specific antibodies in the mouse brain ( d ). For the cropped images, samples were run in the same gels under same experimental conditions and processed in parallel. Each blot is representative for three experiments
    Figure Legend Snippet: Inhibitory effects of MMPP on accumulation of Aβ 1-42 in the brain of LPS-injected mice. ( a ) Aβ accumulation in the brains of LPS-injected mice was determined by thioflavin S staining. The levels of Aβ 1-42 in mice brain ( n = 5) were measured by ELISA ( b ). The activity of β-secretase in mice brain ( n = 5) was investigated using assay kit ( c ). The expression of APP, BACE1 and C99 was detected by Western blotting using specific antibodies in the mouse brain ( d ). For the cropped images, samples were run in the same gels under same experimental conditions and processed in parallel. Each blot is representative for three experiments

    Techniques Used: Injection, Mouse Assay, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Expressing, Western Blot

    Related Articles

    Western Blot:

    Article Title: RACK1 Is a Ribosome Scaffold Protein for ?-actin mRNA/ZBP1 Complex
    Article Snippet: .. For western blotting, proteins were loaded on SDS-PAGE 10% and transferred on PVDF (Millipore) membrane and the following primary antibodies were used: mouse anti-RACK1 (1∶2000), mouse anti-ZBP1 (Huttelmaier 2005, 1∶1000), polyclonal anti-GFP (1∶1000), mouse anti-Actin (Sigma, 1∶1000), polyclonal anti-Src ,polyclonal anti-eIF4E, polyclonal anti-PABP (alls from Cell signalling, 1∶1000) and mouse anti-GAP43 (1∶2000), polyclonal anti-p27BBP /eIF6 (kindly provided by prof. Biffo Stefano, 1∶1000). .. Secondary HRP-coniugated anti-mouse or anti-rabbit antibodies and ECL reagent (GE healthcare) were used.

    Immunohistochemistry:

    Article Title: Receptor for Activated Protein Kinase C: Requirement for Efficient MicroRNA Function and Reduced Expression in Hepatocellular Carcinoma
    Article Snippet: .. An Atlas anti-RACK1 antibody was used in immunohistochemical analyses (HPA021676; Sigma). .. Western blotting Cell extract protein concentrations were measured using a DC Protein Assay Kit (Bio-Rad, Hercules, CA).

    SDS Page:

    Article Title: RACK1 Is a Ribosome Scaffold Protein for ?-actin mRNA/ZBP1 Complex
    Article Snippet: .. For western blotting, proteins were loaded on SDS-PAGE 10% and transferred on PVDF (Millipore) membrane and the following primary antibodies were used: mouse anti-RACK1 (1∶2000), mouse anti-ZBP1 (Huttelmaier 2005, 1∶1000), polyclonal anti-GFP (1∶1000), mouse anti-Actin (Sigma, 1∶1000), polyclonal anti-Src ,polyclonal anti-eIF4E, polyclonal anti-PABP (alls from Cell signalling, 1∶1000) and mouse anti-GAP43 (1∶2000), polyclonal anti-p27BBP /eIF6 (kindly provided by prof. Biffo Stefano, 1∶1000). .. Secondary HRP-coniugated anti-mouse or anti-rabbit antibodies and ECL reagent (GE healthcare) were used.

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    Millipore antibody against dbh
    Anti-dopamine-β-hydroxylase <t>(DBH)-conjugated</t> saporin (DSAP) produces glucocorticoid resistance in the paraventricular nucleus of the hypothalamus (PVH) of high-fat/high-sucrose choice (HFSC)-fed animals. A–G : combined adrenal weights ( A ), thymus weights ( B ), and corticotropin-releasing hormone (CRH) immunoreactivity (ir) in the PVH ( C and D–G ) in mouse IgG-saporin conjugate <t>(MSAP)</t> and DSAP animals given chow or a HFSC diet for 56 days. MGL, mean gray level. H–K : DBH immunoreactivity in the same sections stained for CRH immunoreactivity ( D–G ) in MSAP ( H and J ) and DSAP ( E and G ) animals. Scale bars = 150 μm. dp, PVH dorsal parvicellular part; mpd, PVH dorsal zone of the medial parvicellular part; mpv, PVH ventral zone of the medial parvicellular part; pm, PVH posterior magnocellular part; pv, PVH periventricular part; V3, 3rd ventricle. Values are means ± SE; n = 5 MSAP-chow and DSAP-HFSC and n = 8 DSAP-chow and MSAP-HFSC. ns, Not significant.
    Antibody Against Dbh, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibody against dbh/product/Millipore
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    99
    Millipore mouse anti human β actin monoclonal antibody
    Effect of MSK1-mediated p65 Ser276 phosphorylation in IL-1β-induced SCF expression. A. Human lung fibroblasts in culture were transiently co-transfected with the pGL3e/SCF firefly luciferase construct and a Renilla luciferase construct (pRL-TK) as an internal control. Cells were pre-incubated for 1 h with a combination of SB202190 (SB; 3.5 µM) and PD98059 (PD; 20 µM) or with H89 (10 µM) and treated with IL-1β (20 U/ml). After 150 min, cells were harvested for luciferase activity measurement. The results are expressed as the level of pGL3e/SCF constructions' promoter-driven firefly luciferase expression after correcting for the transfection efficiency by pRL-TK luciferase measurements and represented as a percentage of control values. B. Fibroblasts were transfected with control and anti-MSK1 siRNA (100 nM), or transfection medium alone (control). After 48 hours, inhibition of MSK1 with siRNA was controlled by Western blotting in the cell lysate, using anti-MSK1, with <t>anti-β-actin</t> antibodies as a deposit control. Cells were treated with IL-1β (20 U/ml). SCF protein levels were assessed in the supernatant 5 hours after treatment by ELISA. C . Fibroblasts were transfected with WT or “kinase-dead” (KD) MSK1 plasmid (1 µg), WT or S276C p65 plasmids or transfection medium alone (control), and treated with IL-1β (20 U/ml). SCF protein levels were assessed by ELISA in the supernatant obtained 5 hours after treatment. Results are expressed as percentages of control values of three independent experiments performed in fibroblasts from three different donors.
    Mouse Anti Human β Actin Monoclonal Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore mouse β actin antibody
    Amyloid plaque load and soluble Aβ levels in the brain of APP swe /PS1 in a context of partial MyD88 deficiency . Deposition of Aβ plaques is significantly more abundant in 6 and 9 month-old APP swe /PS1 compared to APP swe /PS1-Myd88 +/- mice (a) . Aβ immunoreactivity in cortex and hippocampus is shown in brain sections of 9 month-old APP swe /PS1 and APP swe /PS1-Myd88 +/- mice. Percentage of area covered by plaques was quantified for mice of 3, 6 and 9 month-old, respectively. n = 9-10. (Two-way ANOVA was performed revealing a significant interaction between factors age and genotype. The comparison of genotype for each age was performed by Student's t -test). To detect soluble Aβ, western blot analysis on 10-20% Tris-Tricine denaturing polyacrylamide gels of extracellular (b) , intracellular (c) and membrane-associated (d) enriched proteins of 6 month-old mice were assessed using monoclonal 6E10 antibody to reveal the different species. Most of Aβ oligomers were significantly higher in the brains of APP swe /PS1-MyD88 +/- (AM) mice than that of APP swe /PS1 (A) in all protein fractions. Bands depicted here were cut from the same membrane for each protein fraction. Values are expressed as optical densities (OD) in arbitrary units (a.u.) of Aβ normalized with <t>β-actin.</t> n = 4-7; Student's t -test; * P
    Mouse β Actin Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Anti-dopamine-β-hydroxylase (DBH)-conjugated saporin (DSAP) produces glucocorticoid resistance in the paraventricular nucleus of the hypothalamus (PVH) of high-fat/high-sucrose choice (HFSC)-fed animals. A–G : combined adrenal weights ( A ), thymus weights ( B ), and corticotropin-releasing hormone (CRH) immunoreactivity (ir) in the PVH ( C and D–G ) in mouse IgG-saporin conjugate (MSAP) and DSAP animals given chow or a HFSC diet for 56 days. MGL, mean gray level. H–K : DBH immunoreactivity in the same sections stained for CRH immunoreactivity ( D–G ) in MSAP ( H and J ) and DSAP ( E and G ) animals. Scale bars = 150 μm. dp, PVH dorsal parvicellular part; mpd, PVH dorsal zone of the medial parvicellular part; mpv, PVH ventral zone of the medial parvicellular part; pm, PVH posterior magnocellular part; pv, PVH periventricular part; V3, 3rd ventricle. Values are means ± SE; n = 5 MSAP-chow and DSAP-HFSC and n = 8 DSAP-chow and MSAP-HFSC. ns, Not significant.

    Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

    Article Title: Obesity, Diabetes and Energy Homeostasis: Catecholaminergic projections into an interconnected forebrain network control the sensitivity of male rats to diet-induced obesity

    doi: 10.1152/ajpregu.00423.2017

    Figure Lengend Snippet: Anti-dopamine-β-hydroxylase (DBH)-conjugated saporin (DSAP) produces glucocorticoid resistance in the paraventricular nucleus of the hypothalamus (PVH) of high-fat/high-sucrose choice (HFSC)-fed animals. A–G : combined adrenal weights ( A ), thymus weights ( B ), and corticotropin-releasing hormone (CRH) immunoreactivity (ir) in the PVH ( C and D–G ) in mouse IgG-saporin conjugate (MSAP) and DSAP animals given chow or a HFSC diet for 56 days. MGL, mean gray level. H–K : DBH immunoreactivity in the same sections stained for CRH immunoreactivity ( D–G ) in MSAP ( H and J ) and DSAP ( E and G ) animals. Scale bars = 150 μm. dp, PVH dorsal parvicellular part; mpd, PVH dorsal zone of the medial parvicellular part; mpv, PVH ventral zone of the medial parvicellular part; pm, PVH posterior magnocellular part; pv, PVH periventricular part; V3, 3rd ventricle. Values are means ± SE; n = 5 MSAP-chow and DSAP-HFSC and n = 8 DSAP-chow and MSAP-HFSC. ns, Not significant.

    Article Snippet: Sections containing the hypothalamus and hindbrains of all MSAP- and DSAP-injected animals were stained using an antibody against DBH (1:10,000 dilution, mouse anti-DBH; catalog no. MAB308, Millipore, Temecula, CA).

    Techniques: Staining

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0004393

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

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

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

    Amyloid plaque load and soluble Aβ levels in the brain of APP swe /PS1 in a context of partial MyD88 deficiency . Deposition of Aβ plaques is significantly more abundant in 6 and 9 month-old APP swe /PS1 compared to APP swe /PS1-Myd88 +/- mice (a) . Aβ immunoreactivity in cortex and hippocampus is shown in brain sections of 9 month-old APP swe /PS1 and APP swe /PS1-Myd88 +/- mice. Percentage of area covered by plaques was quantified for mice of 3, 6 and 9 month-old, respectively. n = 9-10. (Two-way ANOVA was performed revealing a significant interaction between factors age and genotype. The comparison of genotype for each age was performed by Student's t -test). To detect soluble Aβ, western blot analysis on 10-20% Tris-Tricine denaturing polyacrylamide gels of extracellular (b) , intracellular (c) and membrane-associated (d) enriched proteins of 6 month-old mice were assessed using monoclonal 6E10 antibody to reveal the different species. Most of Aβ oligomers were significantly higher in the brains of APP swe /PS1-MyD88 +/- (AM) mice than that of APP swe /PS1 (A) in all protein fractions. Bands depicted here were cut from the same membrane for each protein fraction. Values are expressed as optical densities (OD) in arbitrary units (a.u.) of Aβ normalized with β-actin. n = 4-7; Student's t -test; * P

    Journal: Molecular Neurodegeneration

    Article Title: MyD88-adaptor protein acts as a preventive mechanism for memory deficits in a mouse model of Alzheimer's disease

    doi: 10.1186/1750-1326-6-5

    Figure Lengend Snippet: Amyloid plaque load and soluble Aβ levels in the brain of APP swe /PS1 in a context of partial MyD88 deficiency . Deposition of Aβ plaques is significantly more abundant in 6 and 9 month-old APP swe /PS1 compared to APP swe /PS1-Myd88 +/- mice (a) . Aβ immunoreactivity in cortex and hippocampus is shown in brain sections of 9 month-old APP swe /PS1 and APP swe /PS1-Myd88 +/- mice. Percentage of area covered by plaques was quantified for mice of 3, 6 and 9 month-old, respectively. n = 9-10. (Two-way ANOVA was performed revealing a significant interaction between factors age and genotype. The comparison of genotype for each age was performed by Student's t -test). To detect soluble Aβ, western blot analysis on 10-20% Tris-Tricine denaturing polyacrylamide gels of extracellular (b) , intracellular (c) and membrane-associated (d) enriched proteins of 6 month-old mice were assessed using monoclonal 6E10 antibody to reveal the different species. Most of Aβ oligomers were significantly higher in the brains of APP swe /PS1-MyD88 +/- (AM) mice than that of APP swe /PS1 (A) in all protein fractions. Bands depicted here were cut from the same membrane for each protein fraction. Values are expressed as optical densities (OD) in arbitrary units (a.u.) of Aβ normalized with β-actin. n = 4-7; Student's t -test; * P

    Article Snippet: Membranes were stripped in 25 mM glycine-HCl, pH 2.0, containing 1% SDS to allow β-actin revelation using first a mouse β-actin antibody (MAB1501, 1:10 000; Millipore Bioscience Research Reagents) and then a goat anti-mouse peroxidase conjugated secondary antibody (1:10 000; Jackson ImmunoResearch).

    Techniques: Mouse Assay, Western Blot

    Characterization of pS1943 NMHC-IIA antibodies. (A) MDA-MB-231 cells were transduced with lentivirus expressing control (shC) or shRNAs (sh5 and sh7) against human NMHC-IIA. Buffer- or calf intestinal and lambda phosphatase (ClP)-treated immunoblots were blotted for total NMHC-IIA, pS1943 NMHC-IIA (Millipore antibody) or β-actin. (B) Buffer- or CIP-treated immunoblots (total and pS1943 NMHC-IIA - Millipore antibody) of unphosphorylated, S1943-phos- phorylated (CK2) and S1916-phosphorylated (PKC) myosin-IIA rods. (C) Representative immunoblots showing expression of total NMHC-IIA, NMHC-IIB, pS1943 NMHC-IIA (Millipore antibody) or β-actin in a panel of breast cancer cell lines. (D) Immunohistochemistry of MDA-MB-231–3475 orthotopic tumors (a, b) and spontaneous lung metastatic nodules (c, d), showing expression of total NMHC-IIA or pS1943 NMHC-IIA (Cell Signaling Technology antibody), which appears as a brown stain. Scale bar = 100 μm.

    Journal: Experimental cell research

    Article Title: Myosin-IIA heavy chain phosphorylation on SI943 regulates tumor metastasis

    doi: 10.1016/j.yexcr.2018.06.028

    Figure Lengend Snippet: Characterization of pS1943 NMHC-IIA antibodies. (A) MDA-MB-231 cells were transduced with lentivirus expressing control (shC) or shRNAs (sh5 and sh7) against human NMHC-IIA. Buffer- or calf intestinal and lambda phosphatase (ClP)-treated immunoblots were blotted for total NMHC-IIA, pS1943 NMHC-IIA (Millipore antibody) or β-actin. (B) Buffer- or CIP-treated immunoblots (total and pS1943 NMHC-IIA - Millipore antibody) of unphosphorylated, S1943-phos- phorylated (CK2) and S1916-phosphorylated (PKC) myosin-IIA rods. (C) Representative immunoblots showing expression of total NMHC-IIA, NMHC-IIB, pS1943 NMHC-IIA (Millipore antibody) or β-actin in a panel of breast cancer cell lines. (D) Immunohistochemistry of MDA-MB-231–3475 orthotopic tumors (a, b) and spontaneous lung metastatic nodules (c, d), showing expression of total NMHC-IIA or pS1943 NMHC-IIA (Cell Signaling Technology antibody), which appears as a brown stain. Scale bar = 100 μm.

    Article Snippet: The membranes were then simultaneously immunoblotted with a β-actin monoclonal antibody and either the pS1943 myosin-IIA rabbit antibody from Millipore or an antibody that recognizes total myosin-IIA.

    Techniques: Multiple Displacement Amplification, Transduction, Expressing, Western Blot, Immunohistochemistry, Staining