ripa buffer  (Roche)


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    Structured Review

    Roche ripa buffer
    Ebola GP interacts with tetherin. ( A and B ) 293T cells were <t>transfected</t> with the indicated plasmids, and <t>RIPA</t> lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein"

    Article Title: Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

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

    doi: 10.1073/pnas.0811014106

    Ebola GP interacts with tetherin. ( A and B ) 293T cells were transfected with the indicated plasmids, and RIPA lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input
    Figure Legend Snippet: Ebola GP interacts with tetherin. ( A and B ) 293T cells were transfected with the indicated plasmids, and RIPA lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input

    Techniques Used: Transfection, Immunoprecipitation, Western Blot

    2) Product Images from "Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3"

    Article Title: Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0022290

    An intact SMYD3 MYND domain is required for association with N-CoR and for transcriptional repression. (A) N-CoR co-immunoprecipitates with wildtype SMYD3 but not with SMYD3 MYND domain point mutant C49/S. 293T cells were co-transfected with N-CoR, N-terminal myc-tagged SMYD3 constructs indicated, and with empty vector (vector). 48 hours post-transfection, whole cell RIPA lysates (WCL) were prepared. Fractions of the lysates were subjected to anti-N-CoR co-immunoprecipitation and the remaining 50% served as input. Western analysis was performed with anti-myc antibodies. Myc-SMYD1B, previously shown to interact with N-CoR served as a positive control. (B) Schematic of GAL4-DNA binding domain (DBD) and GAL4-fusion constructs for wild type (GAL4-SMYD3) and MYND domain-mutated (GAL4-SMYD3-C49/S) two hybrid transcription assays. X denotes the location of the C49/S mutation. (C) GAL4-SMYD3 but not GAL4-SMYD3-C49/S represses transcription of a GAL4-UAS containing luciferase reporter. 293T cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 µg) together with GAL4-DBD, or with 1 or 2 µg (indicated as 1X or 2X) of GAL4-SMYD3 (black bars) or GAL4-SMYD3-C49/S (red bars). Transfection efficiencies were normalized to co-transfected renilla luciferase, and percent GAL4 activity was determined in relation to GAL4-DBD set at 100%.
    Figure Legend Snippet: An intact SMYD3 MYND domain is required for association with N-CoR and for transcriptional repression. (A) N-CoR co-immunoprecipitates with wildtype SMYD3 but not with SMYD3 MYND domain point mutant C49/S. 293T cells were co-transfected with N-CoR, N-terminal myc-tagged SMYD3 constructs indicated, and with empty vector (vector). 48 hours post-transfection, whole cell RIPA lysates (WCL) were prepared. Fractions of the lysates were subjected to anti-N-CoR co-immunoprecipitation and the remaining 50% served as input. Western analysis was performed with anti-myc antibodies. Myc-SMYD1B, previously shown to interact with N-CoR served as a positive control. (B) Schematic of GAL4-DNA binding domain (DBD) and GAL4-fusion constructs for wild type (GAL4-SMYD3) and MYND domain-mutated (GAL4-SMYD3-C49/S) two hybrid transcription assays. X denotes the location of the C49/S mutation. (C) GAL4-SMYD3 but not GAL4-SMYD3-C49/S represses transcription of a GAL4-UAS containing luciferase reporter. 293T cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 µg) together with GAL4-DBD, or with 1 or 2 µg (indicated as 1X or 2X) of GAL4-SMYD3 (black bars) or GAL4-SMYD3-C49/S (red bars). Transfection efficiencies were normalized to co-transfected renilla luciferase, and percent GAL4 activity was determined in relation to GAL4-DBD set at 100%.

    Techniques Used: Mutagenesis, Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Positive Control, Binding Assay, Luciferase, Activity Assay

    3) Product Images from "Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM"

    Article Title: Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM

    Journal: Journal of Virology

    doi: 10.1128/JVI.02405-15

    Biochemical assessment of the avidity of CDV H-cSLAM interactions. (A to C) Coimmunoprecipitation assays. CDV H FLAG and HA-tagged wt cSLAM, mutant cSLAM, or lion SLAM were coexpressed in Vero cells and subsequently lysed with RIPA buffer 24 h posttransfection.
    Figure Legend Snippet: Biochemical assessment of the avidity of CDV H-cSLAM interactions. (A to C) Coimmunoprecipitation assays. CDV H FLAG and HA-tagged wt cSLAM, mutant cSLAM, or lion SLAM were coexpressed in Vero cells and subsequently lysed with RIPA buffer 24 h posttransfection.

    Techniques Used: Mutagenesis

    4) Product Images from "Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells"

    Article Title: Chromatin-bound CRM1 recruits SET-Nup214 and NPM1c onto HOX clusters causing aberrant HOX expression in leukemia cells

    Journal: eLife

    doi: 10.7554/eLife.46667

    The effect of KPT-330 on gene expression, and binding of CRM1 to HOX regions in MEGAL and HEL cells. ( A-B ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. ( A ) MEGAL and ( B ) HEL cells were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting using anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( C ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in MEGAL and HEL cells treated without or with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( D ) The data in ( C ) were reanalyzed to obtain a ratio by comparing with the values for DMSO-treated samples. Note that low expression genes ( HOXA9 in HEL and HOXB4 in MEGAL) were omitted in ( D ). Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; *p
    Figure Legend Snippet: The effect of KPT-330 on gene expression, and binding of CRM1 to HOX regions in MEGAL and HEL cells. ( A-B ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. ( A ) MEGAL and ( B ) HEL cells were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting using anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( C ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in MEGAL and HEL cells treated without or with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( D ) The data in ( C ) were reanalyzed to obtain a ratio by comparing with the values for DMSO-treated samples. Note that low expression genes ( HOXA9 in HEL and HOXB4 in MEGAL) were omitted in ( D ). Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; *p

    Techniques Used: Expressing, Binding Assay, Cell Culture, Real-time Polymerase Chain Reaction

    Protein expression and CRM1 binding profiles in HL60 cells. ( A ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. LOUCY and HL60 cell lines were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting with anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( B–D ) CRM1 ChIP-seq for HL60 cells was performed as described in Figure 1 . ( B : whole genome; C : whole chromosome 17 and HOXB cluster, D : whole chromosome seven and HOXA cluster).
    Figure Legend Snippet: Protein expression and CRM1 binding profiles in HL60 cells. ( A ) Protein expression of Nup214, SET-Nup214, CRM1, and GAPDH. LOUCY and HL60 cell lines were cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr; cell lysates were prepared using RIPA buffer and analyzed by immunoblotting with anti-Nup214, anti-CRM1, or anti-GAPDH antibodies. ( B–D ) CRM1 ChIP-seq for HL60 cells was performed as described in Figure 1 . ( B : whole genome; C : whole chromosome 17 and HOXB cluster, D : whole chromosome seven and HOXA cluster).

    Techniques Used: Expressing, Binding Assay, Cell Culture, Chromatin Immunoprecipitation

    The effect of KPT-330 on the protein and HOX gene expression levels in OCI-AML3 cells. ( A ) Protein expression of CRM1, NPM1c and GAPDH in OCI-AML3 cells cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. The cell lysates were prepared in RIPA buffer and analyzed by immunoblotting using anti-CRM1, anti-NPM1c, or anti-GAPDH antibodies. ( B ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in OCI-AML3 cells treated with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( C ) The data in ( B ) were reanalyzed to obtain a ratio by comparing the values of the KPT-330-treated samples with the values for DMSO-treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; **p
    Figure Legend Snippet: The effect of KPT-330 on the protein and HOX gene expression levels in OCI-AML3 cells. ( A ) Protein expression of CRM1, NPM1c and GAPDH in OCI-AML3 cells cultured in the absence or presence of DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. The cell lysates were prepared in RIPA buffer and analyzed by immunoblotting using anti-CRM1, anti-NPM1c, or anti-GAPDH antibodies. ( B ) qPCR analysis of HOX cluster genes ( HOXA9 , HOXA11 , HOXB4 , and HOXB9 ) in OCI-AML3 cells treated with DMSO (vehicle control) or KPT-330 (100 nM or 1000 nM) for 24 hr. GAPDH was used as a reference gene. Data are presented as mean values ± SEM of three independent experiments (n = 3). ( C ) The data in ( B ) were reanalyzed to obtain a ratio by comparing the values of the KPT-330-treated samples with the values for DMSO-treated samples. Data are presented as mean values ± SEM. Asterisks indicate statistical significance determined by Student’s t -test; **p

    Techniques Used: Expressing, Cell Culture, Real-time Polymerase Chain Reaction

    5) Product Images from "Dystonia type 6 gene product Thap1: identification of a 50 kDa DNA-binding species in neuronal nuclear fractions"

    Article Title: Dystonia type 6 gene product Thap1: identification of a 50 kDa DNA-binding species in neuronal nuclear fractions

    Journal: Acta Neuropathologica Communications

    doi: 10.1186/s40478-014-0139-1

    A 50 kDa T1-LIR species is detected by Proteintech antibody in mouse neural tissue and testes and is developmentally regulated. (a) Nuclear (150 μg) and cytoplasmic (150 μg) extracts from adult heart, testis, and whole brain were subjected to SDS-PAGE and immunoblotted with Proteintech anti-Thap1 antibody. The 29 kDa T1-LIR species (*) was detected in both compartments in all three tissues. The 32 kDa species (**) was detected only in nuclear extract from testis. The 50 kDa species (arrow) was detected in the nuclear compartment in testis (doublet) and brain, with a trace amount detected in cytoplasm, likely due to contamination with nuclear proteins (not shown). (b) Total cellular extracts (50 μg) from adult peripheral organs (left panel) and adult and E15 individual brain regions (right panel) were immunoblotted with Proteintech anti-Thap1 antibody. “Positive control lysate” lane corresponds to HEK293T cells transfected with a human Thap1 expression vector. (c) Extracts from liver, cerebellum and forebrain from P1 mice were subjected to SDS-PAGE, immunoblotted, and patterns were compared to those observed in adult cerebellum. Total cellular RIPA (150–200 μg), cytoplasmic (cyt) (150–200 μg) and nuclear (nuc) extracts (50–75 μg) were immunoblotted with Proteintech antibody. The lower 50 kDa T1-LIR species (**) was detected only in brain tissues and the levels of the upper 50 kDa (arrow) and lower 50 kDa were increased in P1 relative to adult. Note that the 50+ kDa species in P1 liver nucleus does not co-migrate with either of the two brain species. The 29 kDa species (*) appeared in nuclear extract only in neural tissue.
    Figure Legend Snippet: A 50 kDa T1-LIR species is detected by Proteintech antibody in mouse neural tissue and testes and is developmentally regulated. (a) Nuclear (150 μg) and cytoplasmic (150 μg) extracts from adult heart, testis, and whole brain were subjected to SDS-PAGE and immunoblotted with Proteintech anti-Thap1 antibody. The 29 kDa T1-LIR species (*) was detected in both compartments in all three tissues. The 32 kDa species (**) was detected only in nuclear extract from testis. The 50 kDa species (arrow) was detected in the nuclear compartment in testis (doublet) and brain, with a trace amount detected in cytoplasm, likely due to contamination with nuclear proteins (not shown). (b) Total cellular extracts (50 μg) from adult peripheral organs (left panel) and adult and E15 individual brain regions (right panel) were immunoblotted with Proteintech anti-Thap1 antibody. “Positive control lysate” lane corresponds to HEK293T cells transfected with a human Thap1 expression vector. (c) Extracts from liver, cerebellum and forebrain from P1 mice were subjected to SDS-PAGE, immunoblotted, and patterns were compared to those observed in adult cerebellum. Total cellular RIPA (150–200 μg), cytoplasmic (cyt) (150–200 μg) and nuclear (nuc) extracts (50–75 μg) were immunoblotted with Proteintech antibody. The lower 50 kDa T1-LIR species (**) was detected only in brain tissues and the levels of the upper 50 kDa (arrow) and lower 50 kDa were increased in P1 relative to adult. Note that the 50+ kDa species in P1 liver nucleus does not co-migrate with either of the two brain species. The 29 kDa species (*) appeared in nuclear extract only in neural tissue.

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

    6) Product Images from "P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance"

    Article Title: P2X7 Receptor Regulates Internalization of Antimicrobial Peptide LL-37 by Human Macrophages That Promotes Intracellular Pathogen Clearance

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1402845

    Clathrin- and caveolae/lipid raft–dependent endocytosis pathways are involved in LL-37 internalization by dTHP-1 cells. HMDMs ( n = 4) ( A ) or dTHP-1 cells ( n = 3) ( B ) were pretreated at 37°C for 1 h with the inhibitors of CME (CLP [10 μM], CLQ [10 μM], or dynasore [20 μM]) or the inhibitors of caveolae/lipid raft–dependent endocytosis (nystatin [10 μg/ml] or filipin [10 μM]). Then the cells were incubated with 10 μg/ml of FAM-labeled LL-37 for an additional hour, and MFI of the cells was analyzed by flow cytometry. ( C ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), CLP (10 μM), or CLQ (25 μM) at 37°C for 1 h and then challenged with LL-37 (10 μg/ml) for an additional hour. After three washes with PBS, the cells were lysed in RIPA buffer. LL-37 in cell lysates was detected by Western blots. The blot is one representative of three independent experiments ( upper panel ). Densitometric analysis of Western blots from three experiments ( lower panel ). ( D ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), dynasore (25 μM), or both inhibitors for 1 h. Subsequently, the cells were challenged with FAM–LL-37 (10 μg/ml), and MFI was analyzed by flow cytometry ( n = 3). ( E ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and CT-B, caveolin-1, or clathrin was visualized using a confocal microscope. ( F ) dTHP-1 cells were treated with TAMRA- or FAM-labeled LL-37 at 37°C for 1 h, and the colocalization of LL-37 and endosome, lysosome, or Golgi was visualized using a confocal microscope. The confocal images are representative of at least three experiments. Images in (E a )–(E c ) and (F a )–(F c ) are enlargements of the boxed areas. Scale bars, 10 μm. * p
    Figure Legend Snippet: Clathrin- and caveolae/lipid raft–dependent endocytosis pathways are involved in LL-37 internalization by dTHP-1 cells. HMDMs ( n = 4) ( A ) or dTHP-1 cells ( n = 3) ( B ) were pretreated at 37°C for 1 h with the inhibitors of CME (CLP [10 μM], CLQ [10 μM], or dynasore [20 μM]) or the inhibitors of caveolae/lipid raft–dependent endocytosis (nystatin [10 μg/ml] or filipin [10 μM]). Then the cells were incubated with 10 μg/ml of FAM-labeled LL-37 for an additional hour, and MFI of the cells was analyzed by flow cytometry. ( C ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), CLP (10 μM), or CLQ (25 μM) at 37°C for 1 h and then challenged with LL-37 (10 μg/ml) for an additional hour. After three washes with PBS, the cells were lysed in RIPA buffer. LL-37 in cell lysates was detected by Western blots. The blot is one representative of three independent experiments ( upper panel ). Densitometric analysis of Western blots from three experiments ( lower panel ). ( D ) dTHP-1 cells were pretreated with nystatin (10 μg/ml), dynasore (25 μM), or both inhibitors for 1 h. Subsequently, the cells were challenged with FAM–LL-37 (10 μg/ml), and MFI was analyzed by flow cytometry ( n = 3). ( E ) dTHP-1 cells were treated with FAM-labeled LL-37 at 37°C for 1 h. The colocalization of LL-37 and CT-B, caveolin-1, or clathrin was visualized using a confocal microscope. ( F ) dTHP-1 cells were treated with TAMRA- or FAM-labeled LL-37 at 37°C for 1 h, and the colocalization of LL-37 and endosome, lysosome, or Golgi was visualized using a confocal microscope. The confocal images are representative of at least three experiments. Images in (E a )–(E c ) and (F a )–(F c ) are enlargements of the boxed areas. Scale bars, 10 μm. * p

    Techniques Used: Incubation, Labeling, Flow Cytometry, Cytometry, Western Blot, Microscopy

    Macrophages take up neutrophil-derived cathelicidin. Human PMNs were pretreated with CytoB (10 μM) and CaCl 2 (2 mM) for 5 min at 37°C and then treated with LTB 4 (100 nM) for 1 min at 37°C. Subsequently, the culture medium was collected. ( A ) dTHP-1 cells were cultured for 1 h in fresh medium or in the conditioned medium from PMNs that were treated or not with LTB 4 . ( B ) Alternatively, dTHP-1 cells were treated with nystatin (10 μg/ml), dynasore (25 μM), KN-62 (10 μM), or oxATP (100 μM) for 1 h, followed by incubation at 37°C in conditioned medium for 1 h. ( C ) In another experiment, control and P2X 7 R-KD dTHP-1 cells were cultured in conditioned medium for 1 h. Subsequently, dTHP-1 cells were washed with 1× PBS and lysed by RIPA buffer. The LL-37 level in dTHP-1 cells was detected by Western blot. The image is one representative of three independent experiments.
    Figure Legend Snippet: Macrophages take up neutrophil-derived cathelicidin. Human PMNs were pretreated with CytoB (10 μM) and CaCl 2 (2 mM) for 5 min at 37°C and then treated with LTB 4 (100 nM) for 1 min at 37°C. Subsequently, the culture medium was collected. ( A ) dTHP-1 cells were cultured for 1 h in fresh medium or in the conditioned medium from PMNs that were treated or not with LTB 4 . ( B ) Alternatively, dTHP-1 cells were treated with nystatin (10 μg/ml), dynasore (25 μM), KN-62 (10 μM), or oxATP (100 μM) for 1 h, followed by incubation at 37°C in conditioned medium for 1 h. ( C ) In another experiment, control and P2X 7 R-KD dTHP-1 cells were cultured in conditioned medium for 1 h. Subsequently, dTHP-1 cells were washed with 1× PBS and lysed by RIPA buffer. The LL-37 level in dTHP-1 cells was detected by Western blot. The image is one representative of three independent experiments.

    Techniques Used: Derivative Assay, Cell Culture, Incubation, Western Blot

    7) Product Images from "Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation"

    Article Title: Microvesicles released from hormone-refractory prostate cancer cells facilitate mouse pre-osteoblast differentiation

    Journal: Journal of Molecular Histology

    doi: 10.1007/s10735-012-9415-1

    Electron-microscoipic observations of hormone-refractory-prostate cancer cell and prostate cancer cell-derived microvesicles (PCa-MVs) ( a ), analysis of microvesicle profile from human prostate cancer PC3 cells ( b ) and biochemical study of MVs from PC3 and DU145 cells ( c ). a Both hormone-refractory-PCa cell lines PC3 cells and DU145 cells shed microvesicles from their cell membrane. The black arrowheads indicate PCa-MVs. The right photos of PC3 and DU145 cells show enlarged views of rectangular areas in the left photos. b MVs were isolated from PC3 cells and measured with NTA. For preparation of MVs from PC3 cells, the medium from either source was centrifuged at 1,500× g for 10 min to remove cells and other debris. These supernatants were then centrifuged at 250,000× g for 3 h at 4 °C. The centrifuged-microvesicles were resuspended in serum-free α-MEM and then filtered (0.45 μm). The microvesicle samples after passage through the 1st filter (0.22 μm) of an ExoMir kit were used for analysis. The Nanosight LM10 nanoparticle characterization system (NanoSight, NanoSight Ltd, UK) equipped with a blue laser (638 nm) illumination was used for real-time characterization of the vesicles. The results were presented at the average value of 2 independent experiments. The number of microvesicles (E6 particles/ml) and the size distribution (particle diameter, nm) are shown on the y axis and x axis, respectively. c Western blots of MV-related proteins, i.e., TSG101, CD9, and CD81. MVs were prepared from the culture medium by ultracentrifugation (250,000× g for 3 h, 4 °C). The microvesicles were further filtered (0.45 μm) and then resuspended in RIPA buffer containing the complete protease inhibitor cocktail. Three micrograms protein per lane was loaded. The relative expression levels of the MV-related proteins indicate a specificity for MVs
    Figure Legend Snippet: Electron-microscoipic observations of hormone-refractory-prostate cancer cell and prostate cancer cell-derived microvesicles (PCa-MVs) ( a ), analysis of microvesicle profile from human prostate cancer PC3 cells ( b ) and biochemical study of MVs from PC3 and DU145 cells ( c ). a Both hormone-refractory-PCa cell lines PC3 cells and DU145 cells shed microvesicles from their cell membrane. The black arrowheads indicate PCa-MVs. The right photos of PC3 and DU145 cells show enlarged views of rectangular areas in the left photos. b MVs were isolated from PC3 cells and measured with NTA. For preparation of MVs from PC3 cells, the medium from either source was centrifuged at 1,500× g for 10 min to remove cells and other debris. These supernatants were then centrifuged at 250,000× g for 3 h at 4 °C. The centrifuged-microvesicles were resuspended in serum-free α-MEM and then filtered (0.45 μm). The microvesicle samples after passage through the 1st filter (0.22 μm) of an ExoMir kit were used for analysis. The Nanosight LM10 nanoparticle characterization system (NanoSight, NanoSight Ltd, UK) equipped with a blue laser (638 nm) illumination was used for real-time characterization of the vesicles. The results were presented at the average value of 2 independent experiments. The number of microvesicles (E6 particles/ml) and the size distribution (particle diameter, nm) are shown on the y axis and x axis, respectively. c Western blots of MV-related proteins, i.e., TSG101, CD9, and CD81. MVs were prepared from the culture medium by ultracentrifugation (250,000× g for 3 h, 4 °C). The microvesicles were further filtered (0.45 μm) and then resuspended in RIPA buffer containing the complete protease inhibitor cocktail. Three micrograms protein per lane was loaded. The relative expression levels of the MV-related proteins indicate a specificity for MVs

    Techniques Used: Derivative Assay, Isolation, Western Blot, Protease Inhibitor, Expressing

    8) Product Images from "RanBP2/Nup358 enhances miRNA activity by sumoylating Argonautes"

    Article Title: RanBP2/Nup358 enhances miRNA activity by sumoylating Argonautes

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1009378

    RanBP2 promotes the sumoylation and inhibits the ubiquitination of AGO1. (A) Unmodified and RanBP2-dE3 U2OS cells were co-transfected with plasmids expressing Flag-HA-tagged AGO1 ( FH-AGO1 ), SV5-tagged Ubc9 ( V5-Ubc9 ), and His-tagged SUMO2 ( His6-SUMO2 “+”) or control vector ( His6-SUMO2 “-”). 24 h post-transfection cells were lysed in 6 M Guanidinium-HCl, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni 2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Input lysates were immunoblotted with antibodies against FH-AGO1, V5-Ubc9, RanBP2 and α-tubulin. See S10A Fig for the quantification of His6-SUMO2-FH-AGO1 levels. (B-C) As in (A), except that unmodified and RanBP2-dE3-1 HEK293 cells were transfected with His6-SUMO1 (B) or His6-SUMO2 (C). Antibodies used for immunoblotting were as indicated on the right. See S10B and S10C Fig for the quantification of His6-SUMO1-FH-AGO1 and His6-SUMO2-FH-AGO1 levels. (D) AGO1 was in vitro sumoylated with purified components, with SUMO2, active recombinant human RanBP2 protein (BP2ΔFG) as the SUMO E3-ligase, and recombinant GST-tagged human AGO1 protein (GST-AGO1) or GST as substrates. Equal amounts of SAE1, SUMO2, Ubc9, GST-AGO1 and GST (35 ng) were added to 10 μL reactions, where 1x is estimated to be 5 ng of BP2ΔFG. Negative controls lacking ATP or BP2ΔFG were also shown. After incubation at 30°C for 1 h, the reactions were analyzed by immunoblotting with antibodies indicated. The position of SUMO2-modified AGO1 is indicated and the SUMO2-conjugated BP2ΔFG is represented by the arrowhead. (E) Similar to (A), except that U2OS cells were co-transfected with plasmids for FH-AGO1 , and His-Myc-tagged ubiquitin ( His-Myc-Ub “+”) or control vector ( His-Myc-Ub “-”). 18 h post-transfection, cells were treated with MG132 (10 μM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in 6 M Guanidinium-HCl, and the His-Myc-Ub conjugates were isolated on Nickel beads (“Ni 2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His-Myc-Ub conjugates by immunoblotting for His (IB: His). See S10D Fig for the quantification of His-Myc-Ub-FH-AGO1 levels. (F) Unmodified and RanBP2-dE3 U2OS cells were transfected with plasmids for FH-AGO1 or Flag-HA-tagged yellow fluorescent protein ( FH-EYFP ). 18 h post-transfection, cells were treated with MG132 (10 μM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and FH-AGO1/FH-EYFP and associated proteins were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed by immunoblotting for ubiquitinated proteins (IB: Ub), immunoprecipitated FH-AGO1/FH-EYFP by immunoblotting against HA, and for co-immunoprecipitated RanBP2. Input lysates were immunoblotted for RanBP2, FH-AGO1 and FH-EYFP (IB: HA) and α-tubulin. See S10E Fig for the quantification of (Ub)n-FH-AGO1 levels. (G) As in (F), except that unmodified and RanBP2-dE3 U2OS cells were co-transfected with FH-AGO1 and either V5-Ubc9 to enhance sumoylation or control plasmid. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). Input lysates were immunoblotted for FH-AGO1 (IB: HA), RanBP2, V5-Ubc9, RanGAP1 and α-tubulin. Note that Ubc9 overexpression rescued RanGAP1-sumoylation in RanBP2-dE3 cells and decreased the amount of ubiquitinated FH-AGO1. See S10F Fig for the quantification of (Ub)n-FH-AGO1 levels. (H) As in (A), except that cells were transfected with either FH-AGO1 or FH-AGO1 K400R . Note that FH-AGO1 K400R was no longer sumoylated in a RanBP2-dependent manner. (I) RanBP2-dE3 U2OS cells were co-transfected with plasmids expressing an intron-containing IL6-HA construct ( IL6-1i-HA ), histone 1B-GFP ( H1B-GFP ) and either FH-AGO1 or FH-AGO1 K400R . Cell lysates were collected 24 h post-transfection, separated by SDS-PAGE and immunoblotted with antibodies against HA (IL6-HA, FH-AGO1 and FH-AGO1 K400R ), GFP and α-tubulin. See S11A Fig for the quantification of IL6-HA levels.
    Figure Legend Snippet: RanBP2 promotes the sumoylation and inhibits the ubiquitination of AGO1. (A) Unmodified and RanBP2-dE3 U2OS cells were co-transfected with plasmids expressing Flag-HA-tagged AGO1 ( FH-AGO1 ), SV5-tagged Ubc9 ( V5-Ubc9 ), and His-tagged SUMO2 ( His6-SUMO2 “+”) or control vector ( His6-SUMO2 “-”). 24 h post-transfection cells were lysed in 6 M Guanidinium-HCl, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni 2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Input lysates were immunoblotted with antibodies against FH-AGO1, V5-Ubc9, RanBP2 and α-tubulin. See S10A Fig for the quantification of His6-SUMO2-FH-AGO1 levels. (B-C) As in (A), except that unmodified and RanBP2-dE3-1 HEK293 cells were transfected with His6-SUMO1 (B) or His6-SUMO2 (C). Antibodies used for immunoblotting were as indicated on the right. See S10B and S10C Fig for the quantification of His6-SUMO1-FH-AGO1 and His6-SUMO2-FH-AGO1 levels. (D) AGO1 was in vitro sumoylated with purified components, with SUMO2, active recombinant human RanBP2 protein (BP2ΔFG) as the SUMO E3-ligase, and recombinant GST-tagged human AGO1 protein (GST-AGO1) or GST as substrates. Equal amounts of SAE1, SUMO2, Ubc9, GST-AGO1 and GST (35 ng) were added to 10 μL reactions, where 1x is estimated to be 5 ng of BP2ΔFG. Negative controls lacking ATP or BP2ΔFG were also shown. After incubation at 30°C for 1 h, the reactions were analyzed by immunoblotting with antibodies indicated. The position of SUMO2-modified AGO1 is indicated and the SUMO2-conjugated BP2ΔFG is represented by the arrowhead. (E) Similar to (A), except that U2OS cells were co-transfected with plasmids for FH-AGO1 , and His-Myc-tagged ubiquitin ( His-Myc-Ub “+”) or control vector ( His-Myc-Ub “-”). 18 h post-transfection, cells were treated with MG132 (10 μM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in 6 M Guanidinium-HCl, and the His-Myc-Ub conjugates were isolated on Nickel beads (“Ni 2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His-Myc-Ub conjugates by immunoblotting for His (IB: His). See S10D Fig for the quantification of His-Myc-Ub-FH-AGO1 levels. (F) Unmodified and RanBP2-dE3 U2OS cells were transfected with plasmids for FH-AGO1 or Flag-HA-tagged yellow fluorescent protein ( FH-EYFP ). 18 h post-transfection, cells were treated with MG132 (10 μM) for an additional 7 h to preserve ubiquitinated conjugates. Cells were lysed in RIPA buffer, and FH-AGO1/FH-EYFP and associated proteins were isolated by immunoprecipitation using anti-HA antibodies (“IP HA”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. The immuoprecipitates were analyzed by immunoblotting for ubiquitinated proteins (IB: Ub), immunoprecipitated FH-AGO1/FH-EYFP by immunoblotting against HA, and for co-immunoprecipitated RanBP2. Input lysates were immunoblotted for RanBP2, FH-AGO1 and FH-EYFP (IB: HA) and α-tubulin. See S10E Fig for the quantification of (Ub)n-FH-AGO1 levels. (G) As in (F), except that unmodified and RanBP2-dE3 U2OS cells were co-transfected with FH-AGO1 and either V5-Ubc9 to enhance sumoylation or control plasmid. The immuoprecipitates were analyzed for ubiquitinated proteins by immunoblotting against ubiquitin (IB: Ub). Input lysates were immunoblotted for FH-AGO1 (IB: HA), RanBP2, V5-Ubc9, RanGAP1 and α-tubulin. Note that Ubc9 overexpression rescued RanGAP1-sumoylation in RanBP2-dE3 cells and decreased the amount of ubiquitinated FH-AGO1. See S10F Fig for the quantification of (Ub)n-FH-AGO1 levels. (H) As in (A), except that cells were transfected with either FH-AGO1 or FH-AGO1 K400R . Note that FH-AGO1 K400R was no longer sumoylated in a RanBP2-dependent manner. (I) RanBP2-dE3 U2OS cells were co-transfected with plasmids expressing an intron-containing IL6-HA construct ( IL6-1i-HA ), histone 1B-GFP ( H1B-GFP ) and either FH-AGO1 or FH-AGO1 K400R . Cell lysates were collected 24 h post-transfection, separated by SDS-PAGE and immunoblotted with antibodies against HA (IL6-HA, FH-AGO1 and FH-AGO1 K400R ), GFP and α-tubulin. See S11A Fig for the quantification of IL6-HA levels.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Isolation, SDS Page, In Vitro, Purification, Recombinant, Incubation, Modification, Immunoprecipitation, Over Expression, Construct

    Nuclear Argonautes interact with IL6 mRNA, and require RanBP2-dependent sumoylation to maintain their association with mRNA in the cytosol. (A) U2OS cells were either directly lysed in RIPA buffer (“Input”) or fractionated into cytoplasmic (“Cyto”), ER and nuclear (“Nuc”) fractions. Samples were separated by SDS-PAGE and immunoblotted for AGO1, GW182, α-tubulin (cytosolic marker), Trapα (ER marker) and Aly (nuclear marker). (B) Similar to (A), except that U2OS cells were infected with lentivirus that delivered shRNA3 against RanBP2 for four days, and were then fractionated into cytoplasmic/ER and nuclear fractions. Samples were analyzed by immunoblotting with antibodies against mAb414 (RanBP2 and Nup214), AGO1, α-tubulin (cytosolic marker), Trapα (ER marker) and Aly (nuclear marker). (C-D) U2OS cells were transfected with intronless IL6-HA construct ( IL6-Δi-HA ). 24 h post-transfection, cells were fractionated into cytoplasmic/ER and nuclear fractions. Fractions were either collected (“Input”) or immunoprecipitated with control (C1) or anti-AGO synthetic antibody fragment. Samples were separated by SDS-PAGE and immunoblotted for AGO1, AGO2 (shown in both low and high exposure), α-tubulin, Trapα and Aly (C). The amount of IL6 mRNA in the immunoprecipitates was quantified by RT-qPCR, each bar representing the average of four independent experiments ± SEM (D). (E) U2OS cells were transfected with an intron-containing IL6-HA construct ( IL6-1i-HA ). 24 h after transfection, cells were lysed and immunoprecipitated with control (C1) or anti-AGO synthetic antibody fragment. The amount of IL6 intron in the immunoprecipitates was quantified by RT-qPCR, each bar being the average of five independent experiments ± SEM. (F-G) Similar to (C-D), except for unmodified and RanBP2-E3ins HAP1 cells. Each bar is the average of three independent experiments ± SEM. (H-I) Unmodified and RanBP2-dE3 U2OS cells were co-transfected with plasmids for Flag-HA-tagged AGO1 ( FH-AGO1 ), SV5-tagged Ubc9 ( V5-Ubc9 ), with or without His-tagged SUMO2 ( His6-SUMO2 “+/-”) and an intron-containing IL6-HA construct ( IL6-1i-HA “+/-”). 24 h post-transfection, cells were lysed in 6 M Guanidinium-HCl, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni 2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Input lysates were immunoblotted with antibodies as indicated (H). Isolated His6-SUMO2-FH-AGO1 and α-tubulin levels were quantified using densitometry analysis and the ratio of His6-SUMO2-FH-AGO1/α-tubulin was normalized to unmodified U2OS cells without IL6-1i-HA. Each bar is the average of two independent experiments ± SEM. (J) General model for how RanBP2 regulates the silencing of the IL6 mRNA through the sumoylation of AGO1. *P = 0.01–0.05, **P = 0.001–0.01, ***P
    Figure Legend Snippet: Nuclear Argonautes interact with IL6 mRNA, and require RanBP2-dependent sumoylation to maintain their association with mRNA in the cytosol. (A) U2OS cells were either directly lysed in RIPA buffer (“Input”) or fractionated into cytoplasmic (“Cyto”), ER and nuclear (“Nuc”) fractions. Samples were separated by SDS-PAGE and immunoblotted for AGO1, GW182, α-tubulin (cytosolic marker), Trapα (ER marker) and Aly (nuclear marker). (B) Similar to (A), except that U2OS cells were infected with lentivirus that delivered shRNA3 against RanBP2 for four days, and were then fractionated into cytoplasmic/ER and nuclear fractions. Samples were analyzed by immunoblotting with antibodies against mAb414 (RanBP2 and Nup214), AGO1, α-tubulin (cytosolic marker), Trapα (ER marker) and Aly (nuclear marker). (C-D) U2OS cells were transfected with intronless IL6-HA construct ( IL6-Δi-HA ). 24 h post-transfection, cells were fractionated into cytoplasmic/ER and nuclear fractions. Fractions were either collected (“Input”) or immunoprecipitated with control (C1) or anti-AGO synthetic antibody fragment. Samples were separated by SDS-PAGE and immunoblotted for AGO1, AGO2 (shown in both low and high exposure), α-tubulin, Trapα and Aly (C). The amount of IL6 mRNA in the immunoprecipitates was quantified by RT-qPCR, each bar representing the average of four independent experiments ± SEM (D). (E) U2OS cells were transfected with an intron-containing IL6-HA construct ( IL6-1i-HA ). 24 h after transfection, cells were lysed and immunoprecipitated with control (C1) or anti-AGO synthetic antibody fragment. The amount of IL6 intron in the immunoprecipitates was quantified by RT-qPCR, each bar being the average of five independent experiments ± SEM. (F-G) Similar to (C-D), except for unmodified and RanBP2-E3ins HAP1 cells. Each bar is the average of three independent experiments ± SEM. (H-I) Unmodified and RanBP2-dE3 U2OS cells were co-transfected with plasmids for Flag-HA-tagged AGO1 ( FH-AGO1 ), SV5-tagged Ubc9 ( V5-Ubc9 ), with or without His-tagged SUMO2 ( His6-SUMO2 “+/-”) and an intron-containing IL6-HA construct ( IL6-1i-HA “+/-”). 24 h post-transfection, cells were lysed in 6 M Guanidinium-HCl, and the His6-SUMO2 conjugates were isolated on Nickel beads (“Ni 2+ NTA PD”) or the lysates were directly analyzed (“input”) and separated by SDS-PAGE. Conjugates were analyzed for the presence of FH-AGO1 by immunoblotting for HA (IB: HA), and for total His6-SUMO2 conjugates by immunoblotting for His (IB: His). Input lysates were immunoblotted with antibodies as indicated (H). Isolated His6-SUMO2-FH-AGO1 and α-tubulin levels were quantified using densitometry analysis and the ratio of His6-SUMO2-FH-AGO1/α-tubulin was normalized to unmodified U2OS cells without IL6-1i-HA. Each bar is the average of two independent experiments ± SEM. (J) General model for how RanBP2 regulates the silencing of the IL6 mRNA through the sumoylation of AGO1. *P = 0.01–0.05, **P = 0.001–0.01, ***P

    Techniques Used: SDS Page, Marker, Infection, Transfection, Construct, Immunoprecipitation, Quantitative RT-PCR, Isolation

    9) Product Images from "Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes"

    Article Title: Phagocytosis and LPS alter the maturation state of ?-amyloid precursor protein and induce different A? peptide release signatures in human mononuclear phagocytes

    Journal: Journal of Neuroinflammation

    doi: 10.1186/1742-2094-7-59

    Shifts in full length APP patterns induced by the glycosylation inhibitors tunicamycin and brefeldin A in mononuclear phagocyte cultures . Human mononuclear phagocytes were isolated as indicated and left unstimulated on ultra-low attachment plates for 3 days. 6 h, 4 h, 2 h or 0.5 h prior to the lysis of the cells, tunicamycin or brefeldin A were added in a concentration of 10 μg/ml each. Cells were lysed in RIPA buffer and APP expression was analysed by separation on 7.5% SDS-PAGE, subsequent blotting on PVDF-membranes and staining with the 1E8 monoclonal antibody. Staining of β-actin served as a loading control. The right hand side shows a molecular weight standard. Note the slight shift in molecular weight after brefeldin A treatment and the decrease of band APP1 corresponding to mature APP and the increased amounts of the bands APP2 and APP3 after treatment with Tunicamycin.
    Figure Legend Snippet: Shifts in full length APP patterns induced by the glycosylation inhibitors tunicamycin and brefeldin A in mononuclear phagocyte cultures . Human mononuclear phagocytes were isolated as indicated and left unstimulated on ultra-low attachment plates for 3 days. 6 h, 4 h, 2 h or 0.5 h prior to the lysis of the cells, tunicamycin or brefeldin A were added in a concentration of 10 μg/ml each. Cells were lysed in RIPA buffer and APP expression was analysed by separation on 7.5% SDS-PAGE, subsequent blotting on PVDF-membranes and staining with the 1E8 monoclonal antibody. Staining of β-actin served as a loading control. The right hand side shows a molecular weight standard. Note the slight shift in molecular weight after brefeldin A treatment and the decrease of band APP1 corresponding to mature APP and the increased amounts of the bands APP2 and APP3 after treatment with Tunicamycin.

    Techniques Used: Isolation, Lysis, Concentration Assay, Expressing, SDS Page, Staining, Molecular Weight

    10) Product Images from "Functional small RNAs are generated from select miRNA hairpin loops in flies and mammals"

    Article Title: Functional small RNAs are generated from select miRNA hairpin loops in flies and mammals

    Journal: Genes & Development

    doi: 10.1101/gad.211698.112

    Loading of human miRNA loops to human Ago2. ( A , B ) The indicated miRNAs were overexpressed under the control of the CMV promoter together with myc-tagged human Ago2 in HeLa cells. The myc-Ago2 complex was purified in RIPA buffer. The indicated miRNA loops
    Figure Legend Snippet: Loading of human miRNA loops to human Ago2. ( A , B ) The indicated miRNAs were overexpressed under the control of the CMV promoter together with myc-tagged human Ago2 in HeLa cells. The myc-Ago2 complex was purified in RIPA buffer. The indicated miRNA loops

    Techniques Used: Purification

    11) Product Images from "Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Can Mediate Degradation of the Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1)"

    Article Title: Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Can Mediate Degradation of the Low Density Lipoprotein Receptor-Related Protein 1 (LRP-1)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0064145

    PCSK9 induces degradation of LRP-1. A ) PCSK9 transfection. HEK293 cells were transfected with PCSK9-V5 or empty control pIRES-V5 vector prior to being lysed in 1x RIPA. LDLR and LRP-1 levels were examined by Western blot in these cells, as well as in HepG2 cells stably expressing PCSK9-shRNA. PCSK9 levels were assessed using mAb-V5 in HEK293 cells and an anti-PCSK9 antibody in HepG2 cells. The levels of LDLR and LRP-1 were estimated relative to β-actin. Data are representative of at least three independent experiments. B ) PCSK9 media swap. Conditioned serum-free media collected from HEK293 cells transfected with PCSK9-V5 or empty control pIRES-V5 vector was collected and applied to naive HEK293 or HepG2 cells. The effect of exogenous PCSK9 on LDLR and LRP-1 was assessed by Western blotting with anti-hLDLR and LRP-1 antibodies respectively. Cell-associated PCSK9 was measured using mAb-V5. The relative intensities of LDLR and LRP-1 were normalized to β-actin using NIH ImageJ software. Data are representative of at least three independent experiments.
    Figure Legend Snippet: PCSK9 induces degradation of LRP-1. A ) PCSK9 transfection. HEK293 cells were transfected with PCSK9-V5 or empty control pIRES-V5 vector prior to being lysed in 1x RIPA. LDLR and LRP-1 levels were examined by Western blot in these cells, as well as in HepG2 cells stably expressing PCSK9-shRNA. PCSK9 levels were assessed using mAb-V5 in HEK293 cells and an anti-PCSK9 antibody in HepG2 cells. The levels of LDLR and LRP-1 were estimated relative to β-actin. Data are representative of at least three independent experiments. B ) PCSK9 media swap. Conditioned serum-free media collected from HEK293 cells transfected with PCSK9-V5 or empty control pIRES-V5 vector was collected and applied to naive HEK293 or HepG2 cells. The effect of exogenous PCSK9 on LDLR and LRP-1 was assessed by Western blotting with anti-hLDLR and LRP-1 antibodies respectively. Cell-associated PCSK9 was measured using mAb-V5. The relative intensities of LDLR and LRP-1 were normalized to β-actin using NIH ImageJ software. Data are representative of at least three independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Stable Transfection, Expressing, shRNA, Software

    Independent actions of PCSK9 on the LDLR and LRP-1. A ) PCSK9 acts on LRP-1 independent of the LDLR. CHO-A7 cells were transfected with control empty pIRES vector, PCSK9-V5, LDLR-V5, or both PCSK9-V5 and LDLR-V5. Levels of LRP-1 were measured 24 h after transfection by Western blotting using an anti-LRP-1 antibody. LDLR and PCSK9 were detected using mAb-V5. β-actin levels, as detected by an anti-β-actin antibody, were used to normalize the amounts of LDLR and LRP-1 quantified using NIH ImageJ software. Data are representative of two independent experiments. B ) PCSK9 acts on the LDLR independent of LRP-1. CHO-K1 and CHO 13-5-1 were incubated overnight with conditioned serum-free media collected from HEK293 cells transfected with empty pIRES-V5 vector, PCSK9-V5, or PCSK9 D374Y -V5. The cells were then lysed in 1x RIPA and submitted to Western blotting using the following antibodies: anti-hamster LDLR, anti-LRP-1, mAb-V5 to detect bound PCSK9, and anti-β-actin antibody. Intensities of the LDLR and LRP-1 were normalized to those of β-actin using NIH ImageJ software. Data are representative of three independent experiments.
    Figure Legend Snippet: Independent actions of PCSK9 on the LDLR and LRP-1. A ) PCSK9 acts on LRP-1 independent of the LDLR. CHO-A7 cells were transfected with control empty pIRES vector, PCSK9-V5, LDLR-V5, or both PCSK9-V5 and LDLR-V5. Levels of LRP-1 were measured 24 h after transfection by Western blotting using an anti-LRP-1 antibody. LDLR and PCSK9 were detected using mAb-V5. β-actin levels, as detected by an anti-β-actin antibody, were used to normalize the amounts of LDLR and LRP-1 quantified using NIH ImageJ software. Data are representative of two independent experiments. B ) PCSK9 acts on the LDLR independent of LRP-1. CHO-K1 and CHO 13-5-1 were incubated overnight with conditioned serum-free media collected from HEK293 cells transfected with empty pIRES-V5 vector, PCSK9-V5, or PCSK9 D374Y -V5. The cells were then lysed in 1x RIPA and submitted to Western blotting using the following antibodies: anti-hamster LDLR, anti-LRP-1, mAb-V5 to detect bound PCSK9, and anti-β-actin antibody. Intensities of the LDLR and LRP-1 were normalized to those of β-actin using NIH ImageJ software. Data are representative of three independent experiments.

    Techniques Used: Transfection, Plasmid Preparation, Western Blot, Software, Incubation

    PCSK9 acts on the LDLR independent of the receptor's CT and TMD. A ) Generation of chimeric truncated LDLR-V5 constructs. Schematic representation of the LDLR, LDLR lacking its CT (ΔCT), and ΔCT in which the LDLR TMD was swapped with that of ACE2 (ΔCT TMDace2 ) or VLDLR (ΔCT TMDvldlr ). All constructs contained a C-terminal V5-tag. B ) Expression in HEK293 cells. WT and chimeric LDLR constructs were transfected in HEK293 cells. Construct expression was assessed by immunoblotting with mAb-V5. Both mature and immature forms of the LDLR were detected. β-actin was used as a loading control. C) PCSK9 induces LDLR degradation independent of the LDLR's CT and TMD. LDLR, ΔCT, and the ΔCT TMDace2 and ΔCT TMDvldlr chimeric constructs were expressed in HEK293 cells. Twenty-four hours post-transfection, the cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media, which contains both full length PCSK9 and its furin cleaved product at Arg 218 , PCSK9-ΔN 218 [33] . Cells were lysed in 1x RIPA and subjected to Western blot analysis. LDLR and PCSK9 were detected with mAb-V5. β-actin was employed as a loading control. The ability of PCSK9 to induce degradation of the LDLR constructs was quantified using NIH ImageJ software and calculated relative to treatment with pIRES conditioned media. Data are representative of at least three independent experiments. D ) PCSK9 reduces cell surface LDLR levels independent of the receptor's CT and TMD. To assess the ability of PCSK9 added exogenously to HEK293 cells expressing the LDLR or its chimeric constructs, transfected cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media. Subsequently, surface LDLR was quantified by FACS analysis. The values obtained after treatment with PCSK9 are represented graphically relative to treatment with control pIRES. Data are representative of at least three independent experiments. Error bars represent SEM. *, p
    Figure Legend Snippet: PCSK9 acts on the LDLR independent of the receptor's CT and TMD. A ) Generation of chimeric truncated LDLR-V5 constructs. Schematic representation of the LDLR, LDLR lacking its CT (ΔCT), and ΔCT in which the LDLR TMD was swapped with that of ACE2 (ΔCT TMDace2 ) or VLDLR (ΔCT TMDvldlr ). All constructs contained a C-terminal V5-tag. B ) Expression in HEK293 cells. WT and chimeric LDLR constructs were transfected in HEK293 cells. Construct expression was assessed by immunoblotting with mAb-V5. Both mature and immature forms of the LDLR were detected. β-actin was used as a loading control. C) PCSK9 induces LDLR degradation independent of the LDLR's CT and TMD. LDLR, ΔCT, and the ΔCT TMDace2 and ΔCT TMDvldlr chimeric constructs were expressed in HEK293 cells. Twenty-four hours post-transfection, the cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media, which contains both full length PCSK9 and its furin cleaved product at Arg 218 , PCSK9-ΔN 218 [33] . Cells were lysed in 1x RIPA and subjected to Western blot analysis. LDLR and PCSK9 were detected with mAb-V5. β-actin was employed as a loading control. The ability of PCSK9 to induce degradation of the LDLR constructs was quantified using NIH ImageJ software and calculated relative to treatment with pIRES conditioned media. Data are representative of at least three independent experiments. D ) PCSK9 reduces cell surface LDLR levels independent of the receptor's CT and TMD. To assess the ability of PCSK9 added exogenously to HEK293 cells expressing the LDLR or its chimeric constructs, transfected cells were treated overnight with empty vector control pIRES-V5 or PCSK9-V5 conditioned media. Subsequently, surface LDLR was quantified by FACS analysis. The values obtained after treatment with PCSK9 are represented graphically relative to treatment with control pIRES. Data are representative of at least three independent experiments. Error bars represent SEM. *, p

    Techniques Used: Construct, Expressing, Transfection, Plasmid Preparation, Western Blot, Software, FACS

    12) Product Images from "The Development and Use of Clickable Activity Based Protein Profiling Agents for Protein Arginine Deiminase 4"

    Article Title: The Development and Use of Clickable Activity Based Protein Profiling Agents for Protein Arginine Deiminase 4

    Journal: ACS chemical biology

    doi: 10.1021/cb1003515

    Comparison of labeling methods. (A) Labeling of MCF-7 whole cell extracts with BCA. Extracts, prepared from estrogen stimulated MCF7 cells, were incubated with BCA (1 μM) for 30 min at 37 °C. The labeled proteins were visualized by western blotting using streptavidin conjugated HRP. (B) Post inactivation coupling in live MCF-7 cells. Live estrogen stimulated MCF-7 cells were treated with Cl-amidine-YNE (100 μM) for 60 min. Cells were lysed in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) on ice for 10 min. The samples were denatured at 95 °C for 10 min prior to the addition of the TEV-biotin-azide reporter tag (20 μM). The ‘click’ reaction was then initiated by the addition of TCEP (2.5 mM), ligand (0.119 mM final), and CuSO 4 (5 mM final). The labeled proteins were visualized by western blotting using streptavidin conjugated HRP.
    Figure Legend Snippet: Comparison of labeling methods. (A) Labeling of MCF-7 whole cell extracts with BCA. Extracts, prepared from estrogen stimulated MCF7 cells, were incubated with BCA (1 μM) for 30 min at 37 °C. The labeled proteins were visualized by western blotting using streptavidin conjugated HRP. (B) Post inactivation coupling in live MCF-7 cells. Live estrogen stimulated MCF-7 cells were treated with Cl-amidine-YNE (100 μM) for 60 min. Cells were lysed in RIPA buffer (25 mM Tris HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) on ice for 10 min. The samples were denatured at 95 °C for 10 min prior to the addition of the TEV-biotin-azide reporter tag (20 μM). The ‘click’ reaction was then initiated by the addition of TCEP (2.5 mM), ligand (0.119 mM final), and CuSO 4 (5 mM final). The labeled proteins were visualized by western blotting using streptavidin conjugated HRP.

    Techniques Used: Labeling, BIA-KA, Incubation, Western Blot

    13) Product Images from "Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿"

    Article Title: Deletion of the First Cysteine-Rich Region of the Varicella-Zoster Virus Glycoprotein E Ectodomain Abolishes the gE and gI Interaction and Differentially Affects Cell-Cell Spread and Viral Entry ▿

    Journal:

    doi: 10.1128/JVI.00913-08

    Analysis of gE and gI interaction and glycosylation in melanoma cells. (A) Coimmunoprecipitation of gE and gI in infected cells. Melanoma cells were inoculated with the rOka-ΔCys mutant or rOka control, and lysates were collected in RIPA buffer.
    Figure Legend Snippet: Analysis of gE and gI interaction and glycosylation in melanoma cells. (A) Coimmunoprecipitation of gE and gI in infected cells. Melanoma cells were inoculated with the rOka-ΔCys mutant or rOka control, and lysates were collected in RIPA buffer.

    Techniques Used: Infection, Mutagenesis

    14) Product Images from "Nonlinear mechanics of human mitotic chromosomes"

    Article Title: Nonlinear mechanics of human mitotic chromosomes

    Journal: Nature

    doi: 10.1038/s41586-022-04666-5

    Mechanical properties of TOP2A-depleted chromosomes. a , Stretching curves of control chromosomes (blue) and TOP2A-depleted chromosomes (orange). b , Representative bright-field image of a TOP2A-depleted chromosome and corresponding H2B immunofluorescence image. Scale bar, 4 μm. c , The relative extension of darker and brighter H2B–eGFP regions as a function of relative intensity (grey line and shaded region: mean ± s.e.m.). d , Representative bright-field images of a chromosome in its original buffer containing 80 mM KCl and subsequent images after flushing in buffer containing 480 mM KCl followed by buffer containing 80 mM KCl. e , Force-extension curves of a control and a TOP2A-depleted HCT116 chromosome before, during and after exposure to high-salt buffer (480 mM KCl). Changes in length ( f ) and compliance ( g ) of chromosomes before and after the exposing control and TOP2A-depleted chromosomes to high-salt buffer. Two-sided Wilcoxon rank-sum test, * P
    Figure Legend Snippet: Mechanical properties of TOP2A-depleted chromosomes. a , Stretching curves of control chromosomes (blue) and TOP2A-depleted chromosomes (orange). b , Representative bright-field image of a TOP2A-depleted chromosome and corresponding H2B immunofluorescence image. Scale bar, 4 μm. c , The relative extension of darker and brighter H2B–eGFP regions as a function of relative intensity (grey line and shaded region: mean ± s.e.m.). d , Representative bright-field images of a chromosome in its original buffer containing 80 mM KCl and subsequent images after flushing in buffer containing 480 mM KCl followed by buffer containing 80 mM KCl. e , Force-extension curves of a control and a TOP2A-depleted HCT116 chromosome before, during and after exposure to high-salt buffer (480 mM KCl). Changes in length ( f ) and compliance ( g ) of chromosomes before and after the exposing control and TOP2A-depleted chromosomes to high-salt buffer. Two-sided Wilcoxon rank-sum test, * P

    Techniques Used: Immunofluorescence

    15) Product Images from "Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity"

    Article Title: Characterization of the invariable residue 51 mutations of human immunodeficiency virus type 1 capsid protein on in vitro CA assembly and infectivity

    Journal: Retrovirology

    doi: 10.1186/1742-4690-4-69

    Western blot analysis of transfected HeLa-tat cell and precipitated viruses . HeLa-tat cells were transfected with the plasmids indicated using the non-liposomal transfection reagent. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Particles released into the culture supernatant were also clarified and filtered of cell debris and precipitated with Viraffinity (CPG) as recommended by the manufacturer. Denatured cell (A and B) and viral lysates (C) were then separated by SDS-PAGE, transferred onto a nitrocellulose membrane and detected with a rabbit anti-HIV glycoprotein (A), a pool of anti-CAp24 and anti-calnexin (B), and anti-CAp24 (C) antibodies. The positions of specific viral proteins are indicated to the left and the numbers to the right depict positions of molecular mass markers (in kDa). NT, a mock control; WT, wild type; and D51N, D51E, and D51Q are the three CAp24 mutants.
    Figure Legend Snippet: Western blot analysis of transfected HeLa-tat cell and precipitated viruses . HeLa-tat cells were transfected with the plasmids indicated using the non-liposomal transfection reagent. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Particles released into the culture supernatant were also clarified and filtered of cell debris and precipitated with Viraffinity (CPG) as recommended by the manufacturer. Denatured cell (A and B) and viral lysates (C) were then separated by SDS-PAGE, transferred onto a nitrocellulose membrane and detected with a rabbit anti-HIV glycoprotein (A), a pool of anti-CAp24 and anti-calnexin (B), and anti-CAp24 (C) antibodies. The positions of specific viral proteins are indicated to the left and the numbers to the right depict positions of molecular mass markers (in kDa). NT, a mock control; WT, wild type; and D51N, D51E, and D51Q are the three CAp24 mutants.

    Techniques Used: Western Blot, Transfection, SDS Page

    Western blot analysis of cell-type dependent expression of HIV-1 proteins . HeLa-tat III, 293T and COS7 cells were transfected as described above with mutant and wild type proviral DNA constructs. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Denatured cell lysates were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with a pool of two HIV-1 positive sera (A), rabbit anti-cyclophilin A (B), and anti-calnexin (C) antibodies. Positions of specific viral and cellular proteins are indicated on the right.
    Figure Legend Snippet: Western blot analysis of cell-type dependent expression of HIV-1 proteins . HeLa-tat III, 293T and COS7 cells were transfected as described above with mutant and wild type proviral DNA constructs. Forty-eight hrs post-transfection, cells were washed and harvested in 1× RIPA buffer. Denatured cell lysates were then resolved by SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted with a pool of two HIV-1 positive sera (A), rabbit anti-cyclophilin A (B), and anti-calnexin (C) antibodies. Positions of specific viral and cellular proteins are indicated on the right.

    Techniques Used: Western Blot, Expressing, Transfection, Mutagenesis, Construct, SDS Page

    16) Product Images from "Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons"

    Article Title: Role of the transcriptional regulator SP140 in resistance to bacterial infections via repression of type I interferons

    Journal: bioRxiv

    doi: 10.1101/2020.01.07.897553

    Sp110 −/− mice are not susceptible to M. tuberculosis infections. ( A ), BMMs were treated with 10U/ml of IFNγ for 24 hours and cells were lysed with RIPA buffer. 5μg of total protein was loaded on each lane, and immunoblot was performed with respective antibodies as shown. Molecular weight standards are shown on the left of each blot in kDa. Individual membranes were imaged separately. Three independent lines of Sp100 −/− mice were analyzed (denoted lines 61, 65, and 71). ( B-D ), Lung of mice infected with M. tuberculosis were stained with hematoxylin and eosin (H E) for histology ( B ), measured for CFU at 25 days post-infection (Mann-Whitney test) ( C ) or, monitored for survival ( D ). All except B6 mice were bred in-house, and combined results from the three independent Sp110 −/− lines are shown. Representative of 2 experiments ( B , D ); combined results of 3 infections ( C ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005.
    Figure Legend Snippet: Sp110 −/− mice are not susceptible to M. tuberculosis infections. ( A ), BMMs were treated with 10U/ml of IFNγ for 24 hours and cells were lysed with RIPA buffer. 5μg of total protein was loaded on each lane, and immunoblot was performed with respective antibodies as shown. Molecular weight standards are shown on the left of each blot in kDa. Individual membranes were imaged separately. Three independent lines of Sp100 −/− mice were analyzed (denoted lines 61, 65, and 71). ( B-D ), Lung of mice infected with M. tuberculosis were stained with hematoxylin and eosin (H E) for histology ( B ), measured for CFU at 25 days post-infection (Mann-Whitney test) ( C ) or, monitored for survival ( D ). All except B6 mice were bred in-house, and combined results from the three independent Sp110 −/− lines are shown. Representative of 2 experiments ( B , D ); combined results of 3 infections ( C ). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.005.

    Techniques Used: Mouse Assay, Molecular Weight, Infection, Staining, MANN-WHITNEY

    17) Product Images from "Chemoresistance to Cancer Treatment: Benzo-α-Pyrene as Friend or Foe?"

    Article Title: Chemoresistance to Cancer Treatment: Benzo-α-Pyrene as Friend or Foe?

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23040930

    Benzo-α-pyrene reverse the effect of drugs on Akt and MEK-ERK signaling pathways in WHCO1 cells. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, and 10 µM BaP for 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, 5-fluorouracil, and BaP; ( B ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with 5-fluorouracil, paclitaxel, and BaP; ( C ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, paclitaxel, and BaP.
    Figure Legend Snippet: Benzo-α-pyrene reverse the effect of drugs on Akt and MEK-ERK signaling pathways in WHCO1 cells. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, and 10 µM BaP for 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, 5-fluorouracil, and BaP; ( B ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with 5-fluorouracil, paclitaxel, and BaP; ( C ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, paclitaxel, and BaP.

    Techniques Used: BIA-KA

    Benzo-α-pyrene differentially influence the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 in response to chemotherapeutic drugs. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 3.5 µM 5-FU, 4.2 µM cisplatin, 2 µM paclitaxel, and 10 µM BaP for 6, 12, and 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with 5-FU and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( B ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with cisplatin and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( C ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with paclitaxel and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies. GAPDH was used as a loading control.
    Figure Legend Snippet: Benzo-α-pyrene differentially influence the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 in response to chemotherapeutic drugs. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 3.5 µM 5-FU, 4.2 µM cisplatin, 2 µM paclitaxel, and 10 µM BaP for 6, 12, and 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with 5-FU and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( B ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with cisplatin and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( C ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with paclitaxel and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies. GAPDH was used as a loading control.

    Techniques Used: Expressing, BIA-KA

    18) Product Images from "Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic ProteinActivating transcription factor 6 limits intracellular accumulation of mutant alpha("

    Article Title: Quantitative Interactome Proteomics Reveals a Molecular Basis for ATF6-Dependent Regulation of a Destabilized Amyloidogenic ProteinActivating transcription factor 6 limits intracellular accumulation of mutant alpha(

    Journal: bioRxiv

    doi: 10.1101/381525

    A . Amino acid alignment of the germline λ light chain (LC), non-amyloidogenic, energetically-normal LC JTO, and the destabilized, amyloidogenic LC ALLC used in this study. B . Immunoblot of Flag M1 immunopurifications (IP) prepared from HEK293 DAX cells transiently transfected with FT JTO, FT ALLC, untagged ALLC, or mock, as indicated. DSP crosslinking (0.5 mM, x-link) was added to cells prior to lysis where indicated. IPs were washed with either high-detergent RIPA or the more gentle lysis buffer (20 mM Hepes pH 7.5 100 mM NaCl 1% Triton X100), as indicated. Notice that the addition of crosslinker allows IPs to be washed with high-detergent RIPA buffer while retaining interactions with ER proteostasis buffers that are lost in the absence of crosslinking. Lysate inputs are shown as controls. C . Immunoblot of Flag M1 immunopurifications (IP) prepared from HEK293 DAX cells transiently transfected with FT ALLC or mock, as indicated. Cells were crosslinked with the indicated concentration of DSP prior to lysis and IP. Lysate inputs are shown as controls.
    Figure Legend Snippet: A . Amino acid alignment of the germline λ light chain (LC), non-amyloidogenic, energetically-normal LC JTO, and the destabilized, amyloidogenic LC ALLC used in this study. B . Immunoblot of Flag M1 immunopurifications (IP) prepared from HEK293 DAX cells transiently transfected with FT JTO, FT ALLC, untagged ALLC, or mock, as indicated. DSP crosslinking (0.5 mM, x-link) was added to cells prior to lysis where indicated. IPs were washed with either high-detergent RIPA or the more gentle lysis buffer (20 mM Hepes pH 7.5 100 mM NaCl 1% Triton X100), as indicated. Notice that the addition of crosslinker allows IPs to be washed with high-detergent RIPA buffer while retaining interactions with ER proteostasis buffers that are lost in the absence of crosslinking. Lysate inputs are shown as controls. C . Immunoblot of Flag M1 immunopurifications (IP) prepared from HEK293 DAX cells transiently transfected with FT ALLC or mock, as indicated. Cells were crosslinked with the indicated concentration of DSP prior to lysis and IP. Lysate inputs are shown as controls.

    Techniques Used: Transfection, Lysis, Concentration Assay

    19) Product Images from "Keap1 moderates the transcription of virus induced genes through G9a-GLP and NFкB p50 recruitment, and H3K9me2 deposition"

    Article Title: Keap1 moderates the transcription of virus induced genes through G9a-GLP and NFкB p50 recruitment, and H3K9me2 deposition

    Journal: bioRxiv

    doi: 10.1101/2022.02.08.479619

    G9a-GLP inhibitors stabilize Keap1 retention in permeabilized MEFs. (A) Culture with BIX01294 stabilizes Keap1 and NFкB p50 retention in MEFs that are incubated with detergents. The MEFs that are indicated above the lanes were cultured with vehicle (-) or with 20 µM BIX01294 (+) for 48 hours. After culture, the MEFs were incubated sequentially in buffers that contained the detergents indicated above the lanes (see Materials and Methods). The proteins that were released during incubation with each detergent were analyzed by immunoblotting using the antibodies indicated to the left of the images. No proteins were detected in the lanes that were loaded with supernatants from a second incubation with 0.5% Triton X-100 (up arrow in the diagram at the top), and those lanes are not shown. The same samples were analyzed on several membranes as indicated to the right of the images (a, b, c). The images show results from a representative experiment in which Keap1+/+ Nrf2-/- #2 and Keap1-/- Nrf2-/- #4 MEFs were compared. The mobilities of molecular weight markers are indicated to the right of the images. (B) Effects of the concentration of BIX01294 and of the time of culture on Keap1 retention in MEFs. The MEFs that are indicated above the images were cultured with the indicated concentrations (µM) of BIX01294 for 4 h (i, upper panel), and for the indicated times (h) with 20 µM BIX01294 (ii, lower panel). After culture, the MEFs were incubated in RIPA buffer, and the proteins that were retained in the MEFs were analyzed by immunoblotting. The images show αKeap1 C and αVinculin immunoblots of the proteins that were retained in the MEFs. The images show the results from a representative experiment in which Keap1+/+ Nrf2-/- #8 and Keap1-/- Nrf2-/- #5 MEFs were compared. (C) The diagram illustrates the stabilization of Keap1 retention in MEFs by G9a-GLP inhibition.
    Figure Legend Snippet: G9a-GLP inhibitors stabilize Keap1 retention in permeabilized MEFs. (A) Culture with BIX01294 stabilizes Keap1 and NFкB p50 retention in MEFs that are incubated with detergents. The MEFs that are indicated above the lanes were cultured with vehicle (-) or with 20 µM BIX01294 (+) for 48 hours. After culture, the MEFs were incubated sequentially in buffers that contained the detergents indicated above the lanes (see Materials and Methods). The proteins that were released during incubation with each detergent were analyzed by immunoblotting using the antibodies indicated to the left of the images. No proteins were detected in the lanes that were loaded with supernatants from a second incubation with 0.5% Triton X-100 (up arrow in the diagram at the top), and those lanes are not shown. The same samples were analyzed on several membranes as indicated to the right of the images (a, b, c). The images show results from a representative experiment in which Keap1+/+ Nrf2-/- #2 and Keap1-/- Nrf2-/- #4 MEFs were compared. The mobilities of molecular weight markers are indicated to the right of the images. (B) Effects of the concentration of BIX01294 and of the time of culture on Keap1 retention in MEFs. The MEFs that are indicated above the images were cultured with the indicated concentrations (µM) of BIX01294 for 4 h (i, upper panel), and for the indicated times (h) with 20 µM BIX01294 (ii, lower panel). After culture, the MEFs were incubated in RIPA buffer, and the proteins that were retained in the MEFs were analyzed by immunoblotting. The images show αKeap1 C and αVinculin immunoblots of the proteins that were retained in the MEFs. The images show the results from a representative experiment in which Keap1+/+ Nrf2-/- #8 and Keap1-/- Nrf2-/- #5 MEFs were compared. (C) The diagram illustrates the stabilization of Keap1 retention in MEFs by G9a-GLP inhibition.

    Techniques Used: Incubation, Cell Culture, Molecular Weight, Concentration Assay, Western Blot, Inhibition

    20) Product Images from "Apoptosis of cancer cells is triggered by selective crosslinking and inhibition of receptor tyrosine kinases"

    Article Title: Apoptosis of cancer cells is triggered by selective crosslinking and inhibition of receptor tyrosine kinases

    Journal: Communications Biology

    doi: 10.1038/s42003-019-0484-5

    Detergent-resistant RTK complex formation by oligomeric ConA. a Hela cells were transfected with hINSR-GFP and treated with ConA for 20 min, and examined under confocal microscopy. Scale bar, 20 µm. b Hela cells were treated with ConA (20 μg/ml) for 20 min and stimulated with 100 nM insulin for 15 min, then lyzed with either RIPA buffer, or directly solubilized in hot Laemmli buffer and examined by western analysis. c Hela cells were pre-treated with EGTA for 1 h and then treated with 1.8 mM CaCl 2 , the level of INSR was measured by western blot. d Hela cells were pre-treated with lipid raft disruptor MβCD for 30 min, the level of INSR was measured by western blot. e Hela cells were treated with ConA (20 μg/ml) for 20 min and lyzed with 1% CHAPS, RIPA, 1% Triton X-100 or 1% SDS separately. The level of INSR in supernatant (S) and precipitate (P) was measured by western blot. f Hela cells were treated with monomeric ConA or tetrameric ConA for 9 h and stimulated with 100 nM insulin for 15 min. The levels of INSR, AKT, FOXO1, Pro/cleaved CASP9 and the phosphorylation levels of INSR, AKT, FOXO1 were measured by western blot. g Annexin V-FITC/PI analysis was done after monomeric ConA or tetrameric ConA treatments. Results were represented as means with standard errors ( n = 3) and analyzed using one-way ANOVA. Values with different letters ( a – f ) in the same column were significantly different ( p
    Figure Legend Snippet: Detergent-resistant RTK complex formation by oligomeric ConA. a Hela cells were transfected with hINSR-GFP and treated with ConA for 20 min, and examined under confocal microscopy. Scale bar, 20 µm. b Hela cells were treated with ConA (20 μg/ml) for 20 min and stimulated with 100 nM insulin for 15 min, then lyzed with either RIPA buffer, or directly solubilized in hot Laemmli buffer and examined by western analysis. c Hela cells were pre-treated with EGTA for 1 h and then treated with 1.8 mM CaCl 2 , the level of INSR was measured by western blot. d Hela cells were pre-treated with lipid raft disruptor MβCD for 30 min, the level of INSR was measured by western blot. e Hela cells were treated with ConA (20 μg/ml) for 20 min and lyzed with 1% CHAPS, RIPA, 1% Triton X-100 or 1% SDS separately. The level of INSR in supernatant (S) and precipitate (P) was measured by western blot. f Hela cells were treated with monomeric ConA or tetrameric ConA for 9 h and stimulated with 100 nM insulin for 15 min. The levels of INSR, AKT, FOXO1, Pro/cleaved CASP9 and the phosphorylation levels of INSR, AKT, FOXO1 were measured by western blot. g Annexin V-FITC/PI analysis was done after monomeric ConA or tetrameric ConA treatments. Results were represented as means with standard errors ( n = 3) and analyzed using one-way ANOVA. Values with different letters ( a – f ) in the same column were significantly different ( p

    Techniques Used: Transfection, Confocal Microscopy, Western Blot

    21) Product Images from "High-density lipoproteins suppress Aβ-induced PBMC adhesion to human endothelial cells in bioengineered vessels and in monoculture"

    Article Title: High-density lipoproteins suppress Aβ-induced PBMC adhesion to human endothelial cells in bioengineered vessels and in monoculture

    Journal: Molecular Neurodegeneration

    doi: 10.1186/s13024-017-0201-0

    HDL reduces Aβ association, binding and uptake to hCMEC/D3 whereas blocking Aβ binding or uptake reduces PBMC adhesion to hCMEC/D3. a-d hCMEC/D3 were pre-treated with 100 μg/mL of HDL and 0.1 μM a,c Aβ40 or b,d Aβ42 monomers as described in Fig. 2 at either 37 °C (association a,b ) or at 4 °C (binding c,d ). Cells were lysed in RIPA buffer and Aβ were measured using commercial ELISA. e hCMEC/D3 were pre-treated with HDL (1 mg/mL) for 2 h before stimulating with 1 mM of fluorescently labelled Aβ40 or Aβ42 monomers. Scale bar represents 10 μm. f-n hCMEC/D3 were pre-treated with ( f-h ) RAGE blocking antibody, i-k RAP or l-n heparin or heparinase III 60 min before stimulation with Aβ40 or Aβ42 monomers or TNF-α for 3 h. PBMC adhesion assays were conducted as described in Fig. 2 . Graphs represent means ± SD relative to vehicle treated cells for at least 3 independent trials. * p
    Figure Legend Snippet: HDL reduces Aβ association, binding and uptake to hCMEC/D3 whereas blocking Aβ binding or uptake reduces PBMC adhesion to hCMEC/D3. a-d hCMEC/D3 were pre-treated with 100 μg/mL of HDL and 0.1 μM a,c Aβ40 or b,d Aβ42 monomers as described in Fig. 2 at either 37 °C (association a,b ) or at 4 °C (binding c,d ). Cells were lysed in RIPA buffer and Aβ were measured using commercial ELISA. e hCMEC/D3 were pre-treated with HDL (1 mg/mL) for 2 h before stimulating with 1 mM of fluorescently labelled Aβ40 or Aβ42 monomers. Scale bar represents 10 μm. f-n hCMEC/D3 were pre-treated with ( f-h ) RAGE blocking antibody, i-k RAP or l-n heparin or heparinase III 60 min before stimulation with Aβ40 or Aβ42 monomers or TNF-α for 3 h. PBMC adhesion assays were conducted as described in Fig. 2 . Graphs represent means ± SD relative to vehicle treated cells for at least 3 independent trials. * p

    Techniques Used: Binding Assay, Blocking Assay, Enzyme-linked Immunosorbent Assay

    22) Product Images from "The genetic association of the transcription factor NPAT with glycemic response to metformin involves regulation of fuel selection"

    Article Title: The genetic association of the transcription factor NPAT with glycemic response to metformin involves regulation of fuel selection

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0253533

    NPAT detection assay and NPAT cell line development. (A-B) HEK293 cells were transfected with NPAT plasmids for 48 hr prior to cell lysis in Urea lysis buffer. NPAT protein was probed by Western blotting with (A) Ab sc32359 (left panel) or Ab sc32359 plus blocking peptide (right panel), and (B) Ab sc32359 (left panel), Ab 611344 (middle panel) or Ab A30-772A-M (right panel). (C) Soluble cell lysates were prepared in Tris-Triton lysis buffer, IP lysis buffer, RIPA lysis buffer or Urea lysis buffer, and probed with Ab sc32359. (D) Insoluble pellets from C were further lysed in Urea lysis buffer and Western blots probed with same antibodies. (E) Immunofluorescence visualization of recombinant NPAT in HEK293 cells, detected with Ab611344. (F) Stably transfected 293 cells were cultured for 24 hr in the presence of tetracycline (Tet) as indicated. NPAT protein was detected in triplicate samples on Western blots with Ab sc32359 and quantified by densitometry (lower panel). (G) Cells expressing GFP-SH (control), NPAT1-SH or NPAT2-SH (shRNA for NPAT) were lysed and NPAT protein detected on Western blots using Ab sc32359. NPAT was quantified by densitometry (lower panel). Data are show as mean ± SEM, n = 3. One-way ANOVA with post hoc Tukey-Kramer multiple comparisons test, *p
    Figure Legend Snippet: NPAT detection assay and NPAT cell line development. (A-B) HEK293 cells were transfected with NPAT plasmids for 48 hr prior to cell lysis in Urea lysis buffer. NPAT protein was probed by Western blotting with (A) Ab sc32359 (left panel) or Ab sc32359 plus blocking peptide (right panel), and (B) Ab sc32359 (left panel), Ab 611344 (middle panel) or Ab A30-772A-M (right panel). (C) Soluble cell lysates were prepared in Tris-Triton lysis buffer, IP lysis buffer, RIPA lysis buffer or Urea lysis buffer, and probed with Ab sc32359. (D) Insoluble pellets from C were further lysed in Urea lysis buffer and Western blots probed with same antibodies. (E) Immunofluorescence visualization of recombinant NPAT in HEK293 cells, detected with Ab611344. (F) Stably transfected 293 cells were cultured for 24 hr in the presence of tetracycline (Tet) as indicated. NPAT protein was detected in triplicate samples on Western blots with Ab sc32359 and quantified by densitometry (lower panel). (G) Cells expressing GFP-SH (control), NPAT1-SH or NPAT2-SH (shRNA for NPAT) were lysed and NPAT protein detected on Western blots using Ab sc32359. NPAT was quantified by densitometry (lower panel). Data are show as mean ± SEM, n = 3. One-way ANOVA with post hoc Tukey-Kramer multiple comparisons test, *p

    Techniques Used: Detection Assay, Transfection, Lysis, Western Blot, Blocking Assay, Immunofluorescence, Recombinant, Stable Transfection, Cell Culture, Expressing, shRNA

    23) Product Images from "Atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 controls the core epithelial-to-mesenchymal transition-inducing transcription factors"

    Article Title: Atypical ubiquitin E3 ligase complex Skp1-Pam-Fbxo45 controls the core epithelial-to-mesenchymal transition-inducing transcription factors

    Journal: Oncotarget

    doi:

    Functional domains of Fbxo45 for ubiquitination of Zeb2 A. Lysates from 293T cells transfected Flag-Zeb2 with or without HA-Fbxo45 were immunoprecipitated (IP) with anti-Flag M2 antibody, and then immunoblotted. Total lysates were also used as Input for immunoblotting analysis. B. Bacterially expressed GST or GST–Fbxo45, -Fbxo45ΔF-box, -Fbxo45Δmid, or -Fbxo45ΔSPRY fusion proteins and Glutathione-Sepharose beads were incubated with lysates of HeLa cells transfected with Zeb2, Snai1 or Snai2, respectively. The proteins associated with GST–tagged Fbxo45 forms, bound on the Glutathione-Sepharose beads were washed five times with the RIPA buffer before immunoblotting. C. Lysates from 293T cells transfected Flag-Zeb2 with HA-Fbxo45, -Fbxo45Δmid, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were immunoprecipitated with anti-Flag or anti-HA antibodies, and immunoprecipitates were resolved by SDS-PAGE for Western-blot analysis. D. U2OS cells transfected with Flag-Zeb2 and HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were stained using the primary antibodies of anti-Flag M2 or anti-HA, and the second antibodies of Alexa Fluor 568 anti-mouse or Alexa Fluor 488 anti-Rabbit, respectively. Scale: 25μm. E-I. Zeb2 ubiquitination assays by using IP expriments under different conditions: Flag-Zeb2 with or without HA-Ub (E); Flag-Zeb2 with HA-Ub WT, K48R or K63R (F); Flag-Zeb2 and HA-Ub K48-only with or without myc-Fbox45 (G); Flag-Zeb2 and HA-Ub K48-only with siRNA control, siRNAs for Fbxo45 or siRNAs for Pam (H); Flag-Zeb2 and HA-Ub with HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY (I).
    Figure Legend Snippet: Functional domains of Fbxo45 for ubiquitination of Zeb2 A. Lysates from 293T cells transfected Flag-Zeb2 with or without HA-Fbxo45 were immunoprecipitated (IP) with anti-Flag M2 antibody, and then immunoblotted. Total lysates were also used as Input for immunoblotting analysis. B. Bacterially expressed GST or GST–Fbxo45, -Fbxo45ΔF-box, -Fbxo45Δmid, or -Fbxo45ΔSPRY fusion proteins and Glutathione-Sepharose beads were incubated with lysates of HeLa cells transfected with Zeb2, Snai1 or Snai2, respectively. The proteins associated with GST–tagged Fbxo45 forms, bound on the Glutathione-Sepharose beads were washed five times with the RIPA buffer before immunoblotting. C. Lysates from 293T cells transfected Flag-Zeb2 with HA-Fbxo45, -Fbxo45Δmid, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were immunoprecipitated with anti-Flag or anti-HA antibodies, and immunoprecipitates were resolved by SDS-PAGE for Western-blot analysis. D. U2OS cells transfected with Flag-Zeb2 and HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY were stained using the primary antibodies of anti-Flag M2 or anti-HA, and the second antibodies of Alexa Fluor 568 anti-mouse or Alexa Fluor 488 anti-Rabbit, respectively. Scale: 25μm. E-I. Zeb2 ubiquitination assays by using IP expriments under different conditions: Flag-Zeb2 with or without HA-Ub (E); Flag-Zeb2 with HA-Ub WT, K48R or K63R (F); Flag-Zeb2 and HA-Ub K48-only with or without myc-Fbox45 (G); Flag-Zeb2 and HA-Ub K48-only with siRNA control, siRNAs for Fbxo45 or siRNAs for Pam (H); Flag-Zeb2 and HA-Ub with HA-Fbxo45, -Fbxo45ΔF-box or -Fbxo45ΔSPRY (I).

    Techniques Used: Functional Assay, Transfection, Immunoprecipitation, Incubation, SDS Page, Western Blot, Staining

    24) Product Images from "Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins"

    Article Title: Automethylation of SUV39H2, an oncogenic histone lysine methyltransferase, regulates its binding affinity to substrate proteins

    Journal: Oncotarget

    doi: 10.18632/oncotarget.8072

    Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.
    Figure Legend Snippet: Automethylation of SUV39H2 is validated in vivo A. Determination of the titer and specificity of the anti-K392 dimethylated SUV39H2 (Sigma-Aldrich) antibody analyzed by enzyme-linked immunosorbent assay (ELISA). A constant amount of K392 methyl peptide or unmethyl peptide has been coated into the wells of the ELISA, and tested with different dilutions of the antibody. B. His-tagged SUV39H2 recombinant proteins were incubated with or without the cofactor SAM at 30°C for 2 hours. Automethylated SUV39H2 protein was blotted with the anti-SUV39H2 K392me2 antibody, and amounts of loading SUV39H2 recombinant proteins were measured by staining with Coomassie Brilliant Blue. C. In vivo methyltransferase experiment was conducted in 293T cells overexpressing FLAG control empty vector (FLAG-Mock), FLAG-tagged SUV39H2 wild-type (FLAG-SUV39H2-WT), FLAG-tagged SUV39H2 K392A mutant (FLAG-SUV39H2-K392A) or FLAG-tagged SUV39H2 K392R mutant (FLAG-SUV39H2-K293R). Cells were lysed with RIPA buffer 48 hours after transfection, and samples were immunoblotted with anti-FLAG and anti-SUV39H2 K392me2 antibodies.

    Techniques Used: In Vivo, Enzyme-linked Immunosorbent Assay, Recombinant, Incubation, Staining, Plasmid Preparation, Mutagenesis, Transfection

    Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.
    Figure Legend Snippet: Automethylation of SUV39H2 blocks the protein-substrate interaction A. 293T cells were co-expressed with HA-tagged LSD1 and FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. After 48 hours of incubation, cells were lysed with RIPA buffer, followed by immunoprecipitation with anti-FLAG M2 affinity gel. Immunoprecipitates were immunoblotted with anti-FLAG and anti-HA antibodies. B. 293T cells were transfected with FLAG-tagged SUV39H2-WT, SUV39H2-K392A or SUV39H2-K392R. Interaction of endogenous histone H3 and exogenous SUV39H2 proteins was examined by western blot analysis.

    Techniques Used: Incubation, Immunoprecipitation, Transfection, Western Blot

    25) Product Images from "Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma"

    Article Title: Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma

    Journal: Scientific Reports

    doi: 10.1038/srep21596

    USP4 regulated the expression of β-catenin by controlling its protein stability. ( A ) To compare the β-catenin protein stability, PC14PE6 and PC14PE6/LvBr4 cells were treated with cycloheximide (20 μg/mL) and harvested at the indicated times. Whole cell lysates were prepared, and the level of β-catenin protein was determined by western blotting. The stability of β-catenin was assessed by image analysis. ( B ) The expression level of USP4 in PC14PE6 and PC14PE6/LvBr4 cells was examined by western blotting (left panel) and RT-qPCR (right panel). ( C ) To determine whether USP regulates the expression of β-catenin, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The whole cell extract was prepared and the expression level of USP4 and β-catenin was determined by western blotting. ( D,E ) To evaluate the direct interaction between USP4 and β-catenin, whole cell lysates were prepared using RIPA buffer, and equal amounts of protein were incubated with appropriate control IgG or an antibody against USP4 ( D ) or β-catenin ( E ). The level of β-catenin and USP4 in IP materials was assessed by western blotting. ( F ) To check the effect of USP4 silencing on the ubiquitination of β-catenin, PC14PE6 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. Whole cell lysates were prepared using RIPA buffer and incubated with appropriate control IgG and β-catenin antibody. The levels of β-catenin and ubiquitin were assessed by western blotting. ( G ) To compare the protein stability of β-catenin, parental PC14PE6 and brain metastatic PC14PE6/LvBr4 cells were treated with cycloheximide (40 μg/mL) and harvested at the indicated times. The stability of β-catenin was determined as described above. Data are means and standard deviation from more than three independent experiments. * p
    Figure Legend Snippet: USP4 regulated the expression of β-catenin by controlling its protein stability. ( A ) To compare the β-catenin protein stability, PC14PE6 and PC14PE6/LvBr4 cells were treated with cycloheximide (20 μg/mL) and harvested at the indicated times. Whole cell lysates were prepared, and the level of β-catenin protein was determined by western blotting. The stability of β-catenin was assessed by image analysis. ( B ) The expression level of USP4 in PC14PE6 and PC14PE6/LvBr4 cells was examined by western blotting (left panel) and RT-qPCR (right panel). ( C ) To determine whether USP regulates the expression of β-catenin, brain metastatic PC14PE6/LvBr4 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. The whole cell extract was prepared and the expression level of USP4 and β-catenin was determined by western blotting. ( D,E ) To evaluate the direct interaction between USP4 and β-catenin, whole cell lysates were prepared using RIPA buffer, and equal amounts of protein were incubated with appropriate control IgG or an antibody against USP4 ( D ) or β-catenin ( E ). The level of β-catenin and USP4 in IP materials was assessed by western blotting. ( F ) To check the effect of USP4 silencing on the ubiquitination of β-catenin, PC14PE6 cells were transfected with control (CTRL) or USP4-specific siRNA for 48 h. Whole cell lysates were prepared using RIPA buffer and incubated with appropriate control IgG and β-catenin antibody. The levels of β-catenin and ubiquitin were assessed by western blotting. ( G ) To compare the protein stability of β-catenin, parental PC14PE6 and brain metastatic PC14PE6/LvBr4 cells were treated with cycloheximide (40 μg/mL) and harvested at the indicated times. The stability of β-catenin was determined as described above. Data are means and standard deviation from more than three independent experiments. * p

    Techniques Used: Expressing, Western Blot, Quantitative RT-PCR, Transfection, Incubation, Standard Deviation

    26) Product Images from "Gamma-secretase inhibition combined with platinum compounds enhances cell death in a large subset of colorectal cancer cells"

    Article Title: Gamma-secretase inhibition combined with platinum compounds enhances cell death in a large subset of colorectal cancer cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-6-8

    Molecular effects of γ-secretase inhibition on signalling proteins involved in regulating cell growth or death . CCK-81 cells were either left untreated (0), treated with DMSO as a control (DM) or incubated with the γ-secretase inhibitors as indicated. Cells were lysed in RIPA buffer and 50 μg of total protein was subjected to immunoblotting analysis as specified. Both inhibitors result in the down-regulation of Val1744-NICD, which is paralleled by a loss of Hes1 expression, an effect that is evident already after 3 to 6 h. Cleaved PARP, an indicator of cell death, was also analysed and more prominent with L-685,458, which induces cell killing in CCK-81 by an unknown mechanism (Table 1 ). With both GSI, an increase in pErk (pT202pY204) and pAkt (pS473) is evident. In addition, a downregulation of anti-apoptotic Bcl-2 protein was detectable, to a moderate degree by DBZ and more pronounced with L-685458. Actin was analysed as a loading control.
    Figure Legend Snippet: Molecular effects of γ-secretase inhibition on signalling proteins involved in regulating cell growth or death . CCK-81 cells were either left untreated (0), treated with DMSO as a control (DM) or incubated with the γ-secretase inhibitors as indicated. Cells were lysed in RIPA buffer and 50 μg of total protein was subjected to immunoblotting analysis as specified. Both inhibitors result in the down-regulation of Val1744-NICD, which is paralleled by a loss of Hes1 expression, an effect that is evident already after 3 to 6 h. Cleaved PARP, an indicator of cell death, was also analysed and more prominent with L-685,458, which induces cell killing in CCK-81 by an unknown mechanism (Table 1 ). With both GSI, an increase in pErk (pT202pY204) and pAkt (pS473) is evident. In addition, a downregulation of anti-apoptotic Bcl-2 protein was detectable, to a moderate degree by DBZ and more pronounced with L-685458. Actin was analysed as a loading control.

    Techniques Used: Inhibition, Incubation, Expressing

    27) Product Images from "Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane"

    Article Title: Functional Role of the Interaction between Polysialic Acid and Myristoylated Alanine-rich C Kinase Substrate at the Plasma Membrane

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.444034

    Reduction of the level of phosphorylated MARCKS by PSA. A and B , primary hippocampal neurons were incubated without ( control ) or with chondroitin sulfate ( CS ) or colominic acid ( PSA ). A , neurons were lysed with RIPA buffer and probed by Western blot (
    Figure Legend Snippet: Reduction of the level of phosphorylated MARCKS by PSA. A and B , primary hippocampal neurons were incubated without ( control ) or with chondroitin sulfate ( CS ) or colominic acid ( PSA ). A , neurons were lysed with RIPA buffer and probed by Western blot (

    Techniques Used: Incubation, Western Blot

    28) Product Images from "Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells"

    Article Title: Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0071316

    Specific nucleoporins are degraded in HRV16-infected cells. ( A ) Ohio-HeLa cells were infected without (mock) or with HRV16 (MOI of 1) and cells lysed using RIPA buffer containing protease and phosphatase inhibitors at the time points shown. Cell lysates were subjected to SDS-PAGE on 4–20% gradient gels and Western analysis using the indicated primary antibodies/horseradish peroxidise-conjugated secondary antibodies and enhanced chemiluminescence (Perkin Elmer). The specificity of the antibodies is indicated on the left. Bands corresponding to 3C, 3CD’ and 3CD are indicated on the right. p.i. - post-infection. ( B ) Results for densitometric analysis of FG-Nup protein bands (left) and non-FG-Nups (right) such as those shown in (A), where data were normalised to the corresponding values for tubulin, relative to the corresponding values for the mock sample. Densitometric analyses were performed using Image J; values represent the mean (± SD) from two independent experiments.
    Figure Legend Snippet: Specific nucleoporins are degraded in HRV16-infected cells. ( A ) Ohio-HeLa cells were infected without (mock) or with HRV16 (MOI of 1) and cells lysed using RIPA buffer containing protease and phosphatase inhibitors at the time points shown. Cell lysates were subjected to SDS-PAGE on 4–20% gradient gels and Western analysis using the indicated primary antibodies/horseradish peroxidise-conjugated secondary antibodies and enhanced chemiluminescence (Perkin Elmer). The specificity of the antibodies is indicated on the left. Bands corresponding to 3C, 3CD’ and 3CD are indicated on the right. p.i. - post-infection. ( B ) Results for densitometric analysis of FG-Nup protein bands (left) and non-FG-Nups (right) such as those shown in (A), where data were normalised to the corresponding values for tubulin, relative to the corresponding values for the mock sample. Densitometric analyses were performed using Image J; values represent the mean (± SD) from two independent experiments.

    Techniques Used: Infection, SDS Page, Western Blot

    29) Product Images from "Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease"

    Article Title: Exome-wide association study identifies a TM6SF2 variant that confers susceptibility to nonalcoholic fatty liver disease

    Journal: Nature genetics

    doi: 10.1038/ng.2901

    Expression of TM6SF2 in cultured hepatocytes. ( a ) Plasmids encoding wild-type and mutant human TM6SF2 were expressed in HuH7 cells. Two days after transfection, the TM6SF2 mRNA levels were measured using Real-Time PCR (left). The cells were harvested and solubilized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH=8). Quantitative immunoblotting was performed using a LI-COR Odyssey infrared imaging system as described in the Methods (right). The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments. V, vector. ( b ) Recombinant wild-type hTM6SF2 was expressed in Hepa1c1c7 cells. After two days, the cells were fractionated and subjected to immunoblotting as described in the Methods. C, cytosol; M, membranes; LD, lipid droplets; L, whole cell lysate. The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments.
    Figure Legend Snippet: Expression of TM6SF2 in cultured hepatocytes. ( a ) Plasmids encoding wild-type and mutant human TM6SF2 were expressed in HuH7 cells. Two days after transfection, the TM6SF2 mRNA levels were measured using Real-Time PCR (left). The cells were harvested and solubilized in RIPA buffer (150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 50 mM Tris, pH=8). Quantitative immunoblotting was performed using a LI-COR Odyssey infrared imaging system as described in the Methods (right). The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments. V, vector. ( b ) Recombinant wild-type hTM6SF2 was expressed in Hepa1c1c7 cells. After two days, the cells were fractionated and subjected to immunoblotting as described in the Methods. C, cytosol; M, membranes; LD, lipid droplets; L, whole cell lysate. The experiment was performed twice and the results were similar. The blots shown are representative of two independent experiments.

    Techniques Used: Expressing, Cell Culture, Mutagenesis, Transfection, Real-time Polymerase Chain Reaction, Imaging, Plasmid Preparation, Recombinant

    30) Product Images from "c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration"

    Article Title: c-Abl and Src-family kinases cross-talk in regulation of myeloid cell migration

    Journal: FEBS letters

    doi: 10.1016/j.febslet.2009.11.009

    Macrophage migration requires the c-Abl kinase activity. (A–D) BMDM monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in RIPA buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.
    Figure Legend Snippet: Macrophage migration requires the c-Abl kinase activity. (A–D) BMDM monolayers were wounded with the tip of a pipette and, after washing, added with medium supplemented with 100 ng/ml LPA without (A, B) or plus 10 μM imatinib mesylate (STI) (C) or 10 μM PP2 (D). Monolayers were photographed immediately after the wound (A) or after 6 h from wounding (B–D). (E) Cells migrated into the wound were quantified after different time from wounding. (F) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. To detect phosphorylation of SFKs, lysates were separated on SDS/PAGE gels, proteins blotted on nitrocellulose and blots probed with Abs of the indicated specificity (WB). To detect c-Abl tyrosine phosphorylation, BMDM were lysed in RIPA buffer as described in Section 2 and immunoprecipitated (IP) with anti-Abl Abs before blotting. (G) BMDM were incubated with control medium (−) or medium supplemented with 10 μM imatinib mesylate (STI) or 10 μM PP2 for 30 min before lysis. (H) Phase contrast images of cells at the margin of the wound after 2 h are shown. These were taken at 40× magnification with an Olympus IX50 microscope and acquired with an Olympus c-7070 wide zoom Digital compact camera. Inhibitors were used at the concentration reported above. One typical of several experiment performed is reported. (I) Baf3 cells expressing Bcr/Abl are strongly polarized. Baf3 cells transfected with an empty vector (Baf3/pSrl) or a vector containing wild-type Bcr/Abl (Baf3/p210wt) or Bcr-Abl with the T315 mutation (Baf3/T315) were cultivated and incubated with STI (10 μM) or PP2 (10 μM) as described in Section 2. Morphology of cells either untreated or treated with STI or PP2 is shown. Cells were plated in 24 well plates in RPMI 1640 medium containing 10% FBS and phase contrast images at 40× magnification taken as described in the legend.

    Techniques Used: Migration, Activity Assay, Transferring, Incubation, Lysis, SDS Page, Western Blot, Immunoprecipitation, Microscopy, Concentration Assay, Expressing, Transfection, Plasmid Preparation, Mutagenesis

    31) Product Images from "The Impact of Rubella Virus Infection on a Secondary Inflammatory Response in Polarized Human Macrophages"

    Article Title: The Impact of Rubella Virus Infection on a Secondary Inflammatory Response in Polarized Human Macrophages

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2021.772595

    LPS-induced cytokine response after infection with RV and IFN-β. At 24 h after incubation of mock, UV-RV, RV and IFN-β (20 ng/ml) treated MΦ (5x10 5 /ml) the medium was changed and the cells were incubated in the presence and absence of LPS (100 ng/ml). After 6 h TNF-α (A) and IFN-concentrations (B) in the culture supernatants were determined by ELISA and LEGENDPLEX human interferon panel kit, respectively. Data represent means ± SD (n = 3). (C, D) GM-MΦ and M-MΦ (5x10 5 /ml) were incubated with decreasing concentrations of IFN-β (20; 2; 0.2; 0.02 ng/ml) for 24 h. After washing with PBS cells were stimulated with LPS (100 ng/ml) for 6 h. IFN-concentrations were determined by LEGENDPLEX human interferon panel kit. Data represent means ± SD (n = 3). (E, F) GM-MΦ and M-MΦ (5x10 5 /ml) were incubated with increasing concentrations of IFN-β (0.02; 0.2; 2; 20 ng/ml). After 24 h cells were lysed using RIPA buffer and proteins separated by SDS-PAGE were subjected to western blot analysis using antibodies for p-Stat1 and β-actin. One representative blot out of three is shown (E) . (F) Western blot bands were quantified and p-Stat1 normalized to loading control β-actin. Data represent means ± SD (n = 3).
    Figure Legend Snippet: LPS-induced cytokine response after infection with RV and IFN-β. At 24 h after incubation of mock, UV-RV, RV and IFN-β (20 ng/ml) treated MΦ (5x10 5 /ml) the medium was changed and the cells were incubated in the presence and absence of LPS (100 ng/ml). After 6 h TNF-α (A) and IFN-concentrations (B) in the culture supernatants were determined by ELISA and LEGENDPLEX human interferon panel kit, respectively. Data represent means ± SD (n = 3). (C, D) GM-MΦ and M-MΦ (5x10 5 /ml) were incubated with decreasing concentrations of IFN-β (20; 2; 0.2; 0.02 ng/ml) for 24 h. After washing with PBS cells were stimulated with LPS (100 ng/ml) for 6 h. IFN-concentrations were determined by LEGENDPLEX human interferon panel kit. Data represent means ± SD (n = 3). (E, F) GM-MΦ and M-MΦ (5x10 5 /ml) were incubated with increasing concentrations of IFN-β (0.02; 0.2; 2; 20 ng/ml). After 24 h cells were lysed using RIPA buffer and proteins separated by SDS-PAGE were subjected to western blot analysis using antibodies for p-Stat1 and β-actin. One representative blot out of three is shown (E) . (F) Western blot bands were quantified and p-Stat1 normalized to loading control β-actin. Data represent means ± SD (n = 3).

    Techniques Used: Infection, Incubation, Enzyme-linked Immunosorbent Assay, SDS Page, Western Blot

    LPS-induced signal transduction after infection with RV. (A) 24 h after incubation RV-, UV-RV- and mock-infected MΦ (5x10 5 /ml) were incubated in the presence and absence of LPS (100 ng/ml). After 15 min cells were lysed using RIPA buffer and protein fractions were separated by SDS-PAGE and subjected to western blot analysis using Ab specific for the indicated proteins. One representative blot out of three is shown. Western blot bands of mock-, UV-RV- and RV-infected cells incubated without (B) and with LPS (100 ng/ml) (C) were quantified as relative intensities after normalization to the loading control β-actin. Data are presented as percent of optical density of the respective mock control (= 100%) and as means ± SD (n = 3). Statistical analysis was performed using the ANOVA test and significances were calculated to the mock control. *p ≤ 0.05, **p ≤ 0.01.
    Figure Legend Snippet: LPS-induced signal transduction after infection with RV. (A) 24 h after incubation RV-, UV-RV- and mock-infected MΦ (5x10 5 /ml) were incubated in the presence and absence of LPS (100 ng/ml). After 15 min cells were lysed using RIPA buffer and protein fractions were separated by SDS-PAGE and subjected to western blot analysis using Ab specific for the indicated proteins. One representative blot out of three is shown. Western blot bands of mock-, UV-RV- and RV-infected cells incubated without (B) and with LPS (100 ng/ml) (C) were quantified as relative intensities after normalization to the loading control β-actin. Data are presented as percent of optical density of the respective mock control (= 100%) and as means ± SD (n = 3). Statistical analysis was performed using the ANOVA test and significances were calculated to the mock control. *p ≤ 0.05, **p ≤ 0.01.

    Techniques Used: Transduction, Infection, Incubation, SDS Page, Western Blot

    Kinetics of RV replication and RV-induced cytokine response in human MΦs. (A) Immunofluorescence staining of capsid protein (C protein, red), actin (green) and DNA (blue) of RV-infected MΦ and mock controls, 24 hours post infection (hpi). Arrows mark groups of cells arranged in clusters (n = 3). (B) GM-MΦ and M-MΦ (5x10 5 /ml) were infected with RV or ultraviolet light-inactivated RV (RV-UV) and the number of intracellular viral genome copies was determined by quantitative one-step TaqMan PCR at 24 hpi. Data represent means ± SD (n = 3). Statistical analysis was performed using the ANOVA test. (C) Assessment of extracellular virus particles by plaque assays of supernatants of GM-MΦ and M-MΦ collected at indicated time points after infection (n = 3 to 5). Data represent means ± SD. (D) Cells (5x10 5 /ml, 24 hpi) were lysed using RIPA buffer and proteins separated by SDS-PAGE were subjected to western blot analysis using antibodies for the rubella E1 structure protein and β-actin. One representative blot out of three is shown. At 24 hpi (E) secreted TNF-α was determined by ELISA (n = 6, means ± SD) and (F) IFN protein levels by LEGENDPLEX human interferon panel kit (n = 3, means ± SD). Statistical analysis for (E, F) were performed using the ANOVA test. (G) At 12 hpi the medium was changed and the cells were incubated for another 12 h. At 24 hpi the amount of IFNs produced after a medium change (12 hpi) was calculated as fold induction without medium change (= 100%). Data represent means ± SD (n = 3). Statistical analysis was performed using the ANOVA test. At indicated time points the protein expression levels of IFN-α2 (H) , IFN-β (I) and IFN-λ1 (J) were quantified by LEGENDPLEX human interferon panel kit. Data represent means ± SD (n = 3). Student’s t -test and significances were calculated to MΦ treated with RV-UV. IFNLR1 (K) and IFNAR1 (L) mRNA expression were determined by qPCR before RV infection as well as 8 and 24 hpi. Data are presented as relative mRNA expression of pre-infected MΦ (= 1) (n = 3, means ± SD) *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
    Figure Legend Snippet: Kinetics of RV replication and RV-induced cytokine response in human MΦs. (A) Immunofluorescence staining of capsid protein (C protein, red), actin (green) and DNA (blue) of RV-infected MΦ and mock controls, 24 hours post infection (hpi). Arrows mark groups of cells arranged in clusters (n = 3). (B) GM-MΦ and M-MΦ (5x10 5 /ml) were infected with RV or ultraviolet light-inactivated RV (RV-UV) and the number of intracellular viral genome copies was determined by quantitative one-step TaqMan PCR at 24 hpi. Data represent means ± SD (n = 3). Statistical analysis was performed using the ANOVA test. (C) Assessment of extracellular virus particles by plaque assays of supernatants of GM-MΦ and M-MΦ collected at indicated time points after infection (n = 3 to 5). Data represent means ± SD. (D) Cells (5x10 5 /ml, 24 hpi) were lysed using RIPA buffer and proteins separated by SDS-PAGE were subjected to western blot analysis using antibodies for the rubella E1 structure protein and β-actin. One representative blot out of three is shown. At 24 hpi (E) secreted TNF-α was determined by ELISA (n = 6, means ± SD) and (F) IFN protein levels by LEGENDPLEX human interferon panel kit (n = 3, means ± SD). Statistical analysis for (E, F) were performed using the ANOVA test. (G) At 12 hpi the medium was changed and the cells were incubated for another 12 h. At 24 hpi the amount of IFNs produced after a medium change (12 hpi) was calculated as fold induction without medium change (= 100%). Data represent means ± SD (n = 3). Statistical analysis was performed using the ANOVA test. At indicated time points the protein expression levels of IFN-α2 (H) , IFN-β (I) and IFN-λ1 (J) were quantified by LEGENDPLEX human interferon panel kit. Data represent means ± SD (n = 3). Student’s t -test and significances were calculated to MΦ treated with RV-UV. IFNLR1 (K) and IFNAR1 (L) mRNA expression were determined by qPCR before RV infection as well as 8 and 24 hpi. Data are presented as relative mRNA expression of pre-infected MΦ (= 1) (n = 3, means ± SD) *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

    Techniques Used: Immunofluorescence, Staining, Infection, Polymerase Chain Reaction, SDS Page, Western Blot, Enzyme-linked Immunosorbent Assay, Incubation, Produced, Expressing, Real-time Polymerase Chain Reaction

    32) Product Images from "Leishmania donovani Secretory Mevalonate Kinase Regulates Host Immune Response and Facilitates Phagocytosis"

    Article Title: Leishmania donovani Secretory Mevalonate Kinase Regulates Host Immune Response and Facilitates Phagocytosis

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2021.641985

    LdMVK facilitates parasite invasion. (A) rMVK binds to the surface of macrophages. (a) Graph depicts the binding of rLdMVK on macrophages in a dose-dependent manner. Fixed macrophage cells were incubated with increasing protein concentration. The result shows mean of three independent experiments performed in triplicates. (b) Table representing the mean O.D. of experiments performed in triplicates of aforesaid described adhesion assay. (c) Blot representing binding of r-LdMVK to macrophage cell surface. PBMC-derived macrophage was incubated with r-LdMVK for 1 h, washed four times with PBS-CM, and lysed using RIPA buffer containing phosphatase inhibitors. Lysate was separated on SDS-PAGE gel and incubated in anti-MVK antibody. The image is representative of three independent experiments. Treated, macrophages incubated with r-LdMVK; untreated, macrophages without any protein incubation; M, protein marker; last wash, last wash of three washes performed post MVK incubation. (B) Infection of macrophages on rLdMVK treatment. (a) PBMC-derived macrophages were treated with Ld promastigotes, 1 µg/ml r-LdMVK+Ld promastigotes, anti-MVK antibody + Ld promastigotes and r-LdMVK + anti-MVK antibody + Ld promastigotes for 4 (h) Parasite to cell ratio was 10:1. Error bars shows SE from three independent studies. (b) Table depicting the mean value of parasites internalized per 100 macrophages on different incubations. (c) Representative image of Giemsa stained infected macrophages on various treatments. (C) Infection of macrophages by MVK-overexpressed and vector control parasites. (a) Bar graph representing normalized band intensity of exogenous MVK (exogenous-MVK/ β-actin) in VC and MVK-OE parasites. Error bars shows SE from three independent studies. (b) Bar graph representing no significant change in the level of normalized band intensity of endogenous MVK (endogenous-MVK/ β-actin) in VC and MVK-OE parasites. Error bars shows SE from three independent studies. (c) Western blot confirmation of generation of MVK-Overexpression strain. Whole cell lysate of MVK-Overexpression and vector control strains of Ag83 parasites were prepared, separated on SDS-PAGE gel, and probed with anti-MVK antibody. The image is representative of three independent experiments. VC, only pLGFPN transfected parasites; MVK-OE, GFP-tagged MVK-overexpressed parasites. (d) Real time PCR confirmation of generation of MVK-Overexpression strain. RNA from MVK-Overexpression and vector control strains of Ag83 parasites were prepared; after cDNA preparation real time PCR was carried out. Table is showing the mean normalized Ct value from these experiments. (e) Bar graph representing the number of internalized parasites per 100 macrophages. PBMC-derived macrophages were treated with mutant stains of parasites with 1:10 macrophage to parasite ratio for 4 (h) Macrophages were washed, stained, and internalized parasites were counted. The data represents the mean±SE of three experiments carried out in triplicates. (f) Table showing the mean value of internalized parasites per 100 macrophages from three experiments. (g) Representative image of Giemsa stained vector control and MVK overexpressed parasites treated macrophages. p value
    Figure Legend Snippet: LdMVK facilitates parasite invasion. (A) rMVK binds to the surface of macrophages. (a) Graph depicts the binding of rLdMVK on macrophages in a dose-dependent manner. Fixed macrophage cells were incubated with increasing protein concentration. The result shows mean of three independent experiments performed in triplicates. (b) Table representing the mean O.D. of experiments performed in triplicates of aforesaid described adhesion assay. (c) Blot representing binding of r-LdMVK to macrophage cell surface. PBMC-derived macrophage was incubated with r-LdMVK for 1 h, washed four times with PBS-CM, and lysed using RIPA buffer containing phosphatase inhibitors. Lysate was separated on SDS-PAGE gel and incubated in anti-MVK antibody. The image is representative of three independent experiments. Treated, macrophages incubated with r-LdMVK; untreated, macrophages without any protein incubation; M, protein marker; last wash, last wash of three washes performed post MVK incubation. (B) Infection of macrophages on rLdMVK treatment. (a) PBMC-derived macrophages were treated with Ld promastigotes, 1 µg/ml r-LdMVK+Ld promastigotes, anti-MVK antibody + Ld promastigotes and r-LdMVK + anti-MVK antibody + Ld promastigotes for 4 (h) Parasite to cell ratio was 10:1. Error bars shows SE from three independent studies. (b) Table depicting the mean value of parasites internalized per 100 macrophages on different incubations. (c) Representative image of Giemsa stained infected macrophages on various treatments. (C) Infection of macrophages by MVK-overexpressed and vector control parasites. (a) Bar graph representing normalized band intensity of exogenous MVK (exogenous-MVK/ β-actin) in VC and MVK-OE parasites. Error bars shows SE from three independent studies. (b) Bar graph representing no significant change in the level of normalized band intensity of endogenous MVK (endogenous-MVK/ β-actin) in VC and MVK-OE parasites. Error bars shows SE from three independent studies. (c) Western blot confirmation of generation of MVK-Overexpression strain. Whole cell lysate of MVK-Overexpression and vector control strains of Ag83 parasites were prepared, separated on SDS-PAGE gel, and probed with anti-MVK antibody. The image is representative of three independent experiments. VC, only pLGFPN transfected parasites; MVK-OE, GFP-tagged MVK-overexpressed parasites. (d) Real time PCR confirmation of generation of MVK-Overexpression strain. RNA from MVK-Overexpression and vector control strains of Ag83 parasites were prepared; after cDNA preparation real time PCR was carried out. Table is showing the mean normalized Ct value from these experiments. (e) Bar graph representing the number of internalized parasites per 100 macrophages. PBMC-derived macrophages were treated with mutant stains of parasites with 1:10 macrophage to parasite ratio for 4 (h) Macrophages were washed, stained, and internalized parasites were counted. The data represents the mean±SE of three experiments carried out in triplicates. (f) Table showing the mean value of internalized parasites per 100 macrophages from three experiments. (g) Representative image of Giemsa stained vector control and MVK overexpressed parasites treated macrophages. p value

    Techniques Used: Binding Assay, Incubation, Protein Concentration, Cell Adhesion Assay, Derivative Assay, SDS Page, Marker, Infection, Staining, Plasmid Preparation, Western Blot, Over Expression, Transfection, Real-time Polymerase Chain Reaction, Mutagenesis

    33) Product Images from "Activity-dependent ubiquitination of the AMPA receptor subunit GluA2"

    Article Title: Activity-dependent ubiquitination of the AMPA receptor subunit GluA2

    Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

    doi: 10.1523/JNEUROSCI.5944-10.2011

    Neuronal activity specifically promotes GluA2 ubiquitination. A) Cortical neurons were incubated in ACSF before bicuculline was added for 15min. Cells were lysed in RIPA or 1% SDS as described under Materials and Methods. AMPARs were immunoprecipitated using non-immune IgG, GluA1 or GluA2 specific antibodies, and immunoblotted for ubiquitin, GluA1, GluA2 and actin. B) Cortical neurons were incubated in ACSF before bicuculline was added for the indicated time. Cells were lysed, and GluA2 was immunoprecipitated using an anti-GluA2 antibody, and immunoblotted for ubiquitin. C) Cortical neurons were incubated in the absence (−) or presence (+) of bicuculline for 30 min at 37°C in ACSF. Bicuculline was washed and neurons in fresh ACSF were returned to 37°C for recovery periods of 15 or 30 min. Cells were lysed and GluA2 was immunoprecipitated using an anti-GluA2 antibody. Samples were subjected to SDS-PAGE followed by immunoblotted with the indicated antibodies.
    Figure Legend Snippet: Neuronal activity specifically promotes GluA2 ubiquitination. A) Cortical neurons were incubated in ACSF before bicuculline was added for 15min. Cells were lysed in RIPA or 1% SDS as described under Materials and Methods. AMPARs were immunoprecipitated using non-immune IgG, GluA1 or GluA2 specific antibodies, and immunoblotted for ubiquitin, GluA1, GluA2 and actin. B) Cortical neurons were incubated in ACSF before bicuculline was added for the indicated time. Cells were lysed, and GluA2 was immunoprecipitated using an anti-GluA2 antibody, and immunoblotted for ubiquitin. C) Cortical neurons were incubated in the absence (−) or presence (+) of bicuculline for 30 min at 37°C in ACSF. Bicuculline was washed and neurons in fresh ACSF were returned to 37°C for recovery periods of 15 or 30 min. Cells were lysed and GluA2 was immunoprecipitated using an anti-GluA2 antibody. Samples were subjected to SDS-PAGE followed by immunoblotted with the indicated antibodies.

    Techniques Used: Activity Assay, Incubation, Immunoprecipitation, SDS Page

    34) Product Images from "α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis"

    Article Title: α-Viniferin Improves Facial Hyperpigmentation via Accelerating Feedback Termination of cAMP/PKA-Signaled Phosphorylation Circuit in Facultative Melanogenesis

    Journal: Theranostics

    doi: 10.7150/thno.24385

    Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p
    Figure Legend Snippet: Effect of C. sinica or α-viniferin on expression of Tyro gene. B16-F0 cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 48 h (A-C) or 20 h (D, E) in the presence of C. sinica or α-viniferin. Cell lysates were prepared with phosphate buffer, and cell extracts with RIPA buffer. (A) Cell lysates were reacted with 1 mM L-dopa, and the velocity of increasing absorbance values at 475 nm was immediately measured. Tyro activity is represented as the initial velocity of L-dopa oxidation (nmol/min). (B) Cell lysates were resolved on non-denaturing acrylamide gels (without 2-mercaptoethanol) by electrophoresis, and subjected to zymography with soaking of the gels in 1 mM L-dopa. (C) Cell extracts were resolved on SDS-acrylamide gels by electrophoresis, and subjected to Western blot (WB) analysis with anti-Tyro or anti-GAPDH antibody. (D, E) Total RNAs were subjected to RT-PCR analysis of Tyro, TYRP1 or DCT with β-actin as an internal control, and resolved on agarose gels by electrophoresis. (F) B16-F0 cells were transfected with Tyro (-2236/+59)-Luc reporter construct in combination with Renilla control vector. The transfected cells were pretreated with C. sinica or α-viniferin for 2 h and stimulated with α-MSH for 18 h in the presence of C. sinica or α-viniferin. Firefly luciferase activity, a reporter of the promoter activity of Tyro gene, is represented as a relative fold after normalizing to Renilla activity, a reference of transfection efficiency. Data are mean ± SEM. # p

    Techniques Used: Expressing, Activity Assay, Electrophoresis, Zymography, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Luciferase

    35) Product Images from "Ubiquitin-dependent regulation of Cdc42 by XIAP"

    Article Title: Ubiquitin-dependent regulation of Cdc42 by XIAP

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.305

    ( a ) Interaction between XIAP and different Cdc42 mutants was tested via an in vitro GST Pulldown assay. With GST protein as a control, two different concentrations of cleaved Cdc42 mutants were used to test the binding to GST tagged XIAP ( b ) In vitro ubiquitination of Cdc42 by XIAP. Purified recombinant Cdc42Q61L was subjected to in vitro ubiquitination by XIAP and cIAP1 recombinant proteins (protocol described in Materials and Methods). ( c ) HeLa cells were transfected with XIAP siRNA for 48 h and treated with MG132 for 6 h. The cells were lysed in RIPA buffer and His-TUBE immobilized on Ni-NTA beads were employed to enrich the ubiquitinated proteins. The samples were loaded onto a gel and the presence of Cdc42 was monitored by immunoblots. The efficiency of XIAP knockdown was tested in the lysates control. * denotes an unspecific band. ( d ) Gel slices of the in vitro ubiquitination reaction were subjected to mass spectrometric analysis to determine the Lysine(s) responsible for the ubiquitination. Inset shows the gel slices taken for the analysis as well as a comparison between the sequences of Rac1 and Cdc42 ( e ) Mouse embryonic fibroblasts (MEFs) were cultured and lysed to check for Cdc42 levels. Control MEFs and XIAP knockout MEFs stably complemented with different XIAP constructs were used for this experiment. * denotes lower exposure of Cdc42
    Figure Legend Snippet: ( a ) Interaction between XIAP and different Cdc42 mutants was tested via an in vitro GST Pulldown assay. With GST protein as a control, two different concentrations of cleaved Cdc42 mutants were used to test the binding to GST tagged XIAP ( b ) In vitro ubiquitination of Cdc42 by XIAP. Purified recombinant Cdc42Q61L was subjected to in vitro ubiquitination by XIAP and cIAP1 recombinant proteins (protocol described in Materials and Methods). ( c ) HeLa cells were transfected with XIAP siRNA for 48 h and treated with MG132 for 6 h. The cells were lysed in RIPA buffer and His-TUBE immobilized on Ni-NTA beads were employed to enrich the ubiquitinated proteins. The samples were loaded onto a gel and the presence of Cdc42 was monitored by immunoblots. The efficiency of XIAP knockdown was tested in the lysates control. * denotes an unspecific band. ( d ) Gel slices of the in vitro ubiquitination reaction were subjected to mass spectrometric analysis to determine the Lysine(s) responsible for the ubiquitination. Inset shows the gel slices taken for the analysis as well as a comparison between the sequences of Rac1 and Cdc42 ( e ) Mouse embryonic fibroblasts (MEFs) were cultured and lysed to check for Cdc42 levels. Control MEFs and XIAP knockout MEFs stably complemented with different XIAP constructs were used for this experiment. * denotes lower exposure of Cdc42

    Techniques Used: In Vitro, GST Pulldown Assay, Binding Assay, Purification, Recombinant, Transfection, Western Blot, Cell Culture, Knock-Out, Stable Transfection, Construct

    36) Product Images from "Chemoresistance to Cancer Treatment: Benzo-α-Pyrene as Friend or Foe?"

    Article Title: Chemoresistance to Cancer Treatment: Benzo-α-Pyrene as Friend or Foe?

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    doi: 10.3390/molecules23040930

    Benzo-α-pyrene reverse the effect of drugs on Akt and MEK-ERK signaling pathways in WHCO1 cells. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, and 10 µM BaP for 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, 5-fluorouracil, and BaP; ( B ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with 5-fluorouracil, paclitaxel, and BaP; ( C ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, paclitaxel, and BaP.
    Figure Legend Snippet: Benzo-α-pyrene reverse the effect of drugs on Akt and MEK-ERK signaling pathways in WHCO1 cells. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 4.2 µM cisplatin, 3.5 µM 5-FU, 2 µM, and 10 µM BaP for 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, 5-fluorouracil, and BaP; ( B ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with 5-fluorouracil, paclitaxel, and BaP; ( C ) Immunoblot analysis was performed using anti-p-ERK 1, 2, anti-p-Akt 1, 2, anti-ERK2, and anti-Akt2 antibodies after treatment with cisplatin, paclitaxel, and BaP.

    Techniques Used:

    Benzo-α-pyrene differentially influence the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 in response to chemotherapeutic drugs. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 3.5 µM 5-FU, 4.2 µM cisplatin, 2 µM paclitaxel, and 10 µM BaP for 6, 12, and 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with 5-FU and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( B ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with cisplatin and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( C ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with paclitaxel and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies. GAPDH was used as a loading control.
    Figure Legend Snippet: Benzo-α-pyrene differentially influence the expression of CYP1A1, CYP1A2, CYP1B1, and GSTP1 in WHCO1 in response to chemotherapeutic drugs. WHCO1 cells (5 × 10 5 ) were plated in 6-well plates overnight. WHCO1 cells were then treated with 0.1% DMSO, 3.5 µM 5-FU, 4.2 µM cisplatin, 2 µM paclitaxel, and 10 µM BaP for 6, 12, and 24 h. Cells were lysed with RIPA buffer and proteins quantified using the BCA protein quantification assay. ( A ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with 5-FU and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( B ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with cisplatin and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies; ( C ) Immunoblot analysis of proteins extracted from WHCO1 cells treated with paclitaxel and BaP using anti-CYP1A1, CYP1A2, CYP1B1, and GSTP1 antibodies. GAPDH was used as a loading control.

    Techniques Used: Expressing

    37) Product Images from "Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA"

    Article Title: Generation and characterization of an anti-GP73 monoclonal antibody for immunoblotting and sandwich ELISA

    Journal: Journal of Biomedical Research

    doi: 10.7555/JBR.26.20120057

    Characterization of 6A2. A: The specificity of 6A2 was confirmed by Western blotting assays. Lane 1: pre-immune mouse serum, lane 2: purified 6A2, lane 3: final boost mouse serum. B: Immunoprecipitation of 6A2. Lane 1: a positive control (0.2 µg rGP73 protein), lane 2: a negative control (6A2 + RIPA buffer), lane 3: PC-3 cell lysate, lane 4: MCF-7 cell lysate. IHC staining using 6A2 on sections of HCC (C) and BPH (D) tissue (×400). GP73 staining was mainly detected in the perinuclear region of HCC cells and in the luminal side of prostatic gland epithelia (BPH).
    Figure Legend Snippet: Characterization of 6A2. A: The specificity of 6A2 was confirmed by Western blotting assays. Lane 1: pre-immune mouse serum, lane 2: purified 6A2, lane 3: final boost mouse serum. B: Immunoprecipitation of 6A2. Lane 1: a positive control (0.2 µg rGP73 protein), lane 2: a negative control (6A2 + RIPA buffer), lane 3: PC-3 cell lysate, lane 4: MCF-7 cell lysate. IHC staining using 6A2 on sections of HCC (C) and BPH (D) tissue (×400). GP73 staining was mainly detected in the perinuclear region of HCC cells and in the luminal side of prostatic gland epithelia (BPH).

    Techniques Used: Western Blot, Purification, Immunoprecipitation, Positive Control, Negative Control, Immunohistochemistry, Staining

    38) Product Images from "Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly"

    Article Title: Ubiquitination of Ebola virus VP35 at lysine 309 regulates viral transcription and assembly

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1010532

    Lack of ubiquitination and a basic residue at VP35/309 dysregulates virus assembly. ( A ) Lysates from mock or rEBOV-VP35/wt/-K309R or -K309G infected (MOI = 0.01 PFU/cell for 144 hours) VeroE6 cells were immunoprecipitated (IP) with IgG or anti-VP35 antibody with protein A beads in RIPA complete and used for western blot to assess interaction with viral proteins VP40, NP, VP24 and VP30. Lysates used for this experiment were also used for Fig 1C . The area under the curve (AUC) for each protein was calculated using ImageJ from western blots run in triplicate. The relative binding ratio (IP: (viral protein/VP35)/WCE: (viral protein/VP35)) was for all VP35 constructs and divided by wt VP35’s ratio. ( B ) Protein lysates (WCE) from VeroE6 cells infected cells (MOI = 0.01 PFU/cell, 144 hours) with rEBOV-eGFP-VP35/wt (WT), -K309R (R), or -K309G (G) and corresponding sucrose-gradient purified virus. The area under the curve (AUC) for each antibody were calculated using ImageJ from western blots run in triplicate. The packaging ratio (purified virus: (viral protein/VP35)/WCE (panel B): (viral protein/VP35)) was for all VP35 constructs and divided by wt VP35’s ratio. ( C ) The number of viral genome copies was determined using strand-specific qPCR of sucrose-gradient-purified virus. ( D ) The ratio of packaged to intracellular RNA copies was determined using strand-specific qPCR for genomic RNA on lysates from cells and the purified virus, and the ratio was normalized to the value for wt virus. ( E ) The ratio of infectious virus to packaged genome copies was determined by titrating the sucrose-gradient purified virus (PFU/mL) and strand-specific PCR to calculate the vRNA copies in the corresponding sample, and the ratio was normalized to the value for the wt virus. ( F ) The ratio of infectious virus to intracellular genome copies was determined using the supernatant titer and intracellular genome copy number, and the ratio was normalized to the value for the wt virus. This experiment was performed in triplicate ( C-F ). The data analysis was done using a one-way ANOVA with Tukey’s post-test for comparison between groups ( C-F ). P-value: *
    Figure Legend Snippet: Lack of ubiquitination and a basic residue at VP35/309 dysregulates virus assembly. ( A ) Lysates from mock or rEBOV-VP35/wt/-K309R or -K309G infected (MOI = 0.01 PFU/cell for 144 hours) VeroE6 cells were immunoprecipitated (IP) with IgG or anti-VP35 antibody with protein A beads in RIPA complete and used for western blot to assess interaction with viral proteins VP40, NP, VP24 and VP30. Lysates used for this experiment were also used for Fig 1C . The area under the curve (AUC) for each protein was calculated using ImageJ from western blots run in triplicate. The relative binding ratio (IP: (viral protein/VP35)/WCE: (viral protein/VP35)) was for all VP35 constructs and divided by wt VP35’s ratio. ( B ) Protein lysates (WCE) from VeroE6 cells infected cells (MOI = 0.01 PFU/cell, 144 hours) with rEBOV-eGFP-VP35/wt (WT), -K309R (R), or -K309G (G) and corresponding sucrose-gradient purified virus. The area under the curve (AUC) for each antibody were calculated using ImageJ from western blots run in triplicate. The packaging ratio (purified virus: (viral protein/VP35)/WCE (panel B): (viral protein/VP35)) was for all VP35 constructs and divided by wt VP35’s ratio. ( C ) The number of viral genome copies was determined using strand-specific qPCR of sucrose-gradient-purified virus. ( D ) The ratio of packaged to intracellular RNA copies was determined using strand-specific qPCR for genomic RNA on lysates from cells and the purified virus, and the ratio was normalized to the value for wt virus. ( E ) The ratio of infectious virus to packaged genome copies was determined by titrating the sucrose-gradient purified virus (PFU/mL) and strand-specific PCR to calculate the vRNA copies in the corresponding sample, and the ratio was normalized to the value for the wt virus. ( F ) The ratio of infectious virus to intracellular genome copies was determined using the supernatant titer and intracellular genome copy number, and the ratio was normalized to the value for the wt virus. This experiment was performed in triplicate ( C-F ). The data analysis was done using a one-way ANOVA with Tukey’s post-test for comparison between groups ( C-F ). P-value: *

    Techniques Used: Infection, Immunoprecipitation, Western Blot, Binding Assay, Construct, Purification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    The replication of rEBOV-VP35/K309R and -G mutants is attenuated in IFN-competent cells. A549 cells were mock infected (grey) or infected in triplicate wells with rEBOV-eGFP-VP35/wt (black), -K309R (blue), or -K309G (red) at an MOI of 0.01 ( A-C ) or 2.5 PFU/cell ( D-F; J-K ). At different time points, supernatants were collected for virus titration ( A, D ) or for IFNβ ELISA ( I , 48 hpi). The limit of detection (LOD) for the titrations (10 PFU/mL) ( A and D ) and IFNβ (50 pg/mL) ( I ) is indicated (black line). Cells were lysed in either TRIzol for RNA analysis ( B, E, H, J ) or in Laemmli buffer for immunoblot analysis ( K ). qPCR for EBOV RNA ( B and D ), IFNβ mRNA ( H ) or ISG mRNA (J) is shown. The fluorescence microscopy images (GFP) are representative of the three images taken ( C and F ). The difference in titer (log 10 ) between the mutant and wt viruses at the time point corresponding to the wt peak titer is summarized ( G ). The area under the curve (AUC) for each protein was calculated using ImageJ to determine the relative activation of the interferon pathway regulators TBK1, IRF3, and STAT1 (phosphorylated protein/(respective total protein/tubulin)) was normalized to the activation levels in wt-infected cells. The western blots are representative of two independent experiments run in duplicate or triplicate ( K ). The titration ( A and D ), qRT-PCR ( B, D, H and J ), and ELISA ( I ) were done in biological triplicate and are representative of two independent experiments. The data analysis was done using a two-way ANOVA ( A, B, D, E, H, and J ) or one-way ANOVA ( I ) with Bonferroni’s or Tukey’s post-test for comparison between groups, respectively. P-value: *
    Figure Legend Snippet: The replication of rEBOV-VP35/K309R and -G mutants is attenuated in IFN-competent cells. A549 cells were mock infected (grey) or infected in triplicate wells with rEBOV-eGFP-VP35/wt (black), -K309R (blue), or -K309G (red) at an MOI of 0.01 ( A-C ) or 2.5 PFU/cell ( D-F; J-K ). At different time points, supernatants were collected for virus titration ( A, D ) or for IFNβ ELISA ( I , 48 hpi). The limit of detection (LOD) for the titrations (10 PFU/mL) ( A and D ) and IFNβ (50 pg/mL) ( I ) is indicated (black line). Cells were lysed in either TRIzol for RNA analysis ( B, E, H, J ) or in Laemmli buffer for immunoblot analysis ( K ). qPCR for EBOV RNA ( B and D ), IFNβ mRNA ( H ) or ISG mRNA (J) is shown. The fluorescence microscopy images (GFP) are representative of the three images taken ( C and F ). The difference in titer (log 10 ) between the mutant and wt viruses at the time point corresponding to the wt peak titer is summarized ( G ). The area under the curve (AUC) for each protein was calculated using ImageJ to determine the relative activation of the interferon pathway regulators TBK1, IRF3, and STAT1 (phosphorylated protein/(respective total protein/tubulin)) was normalized to the activation levels in wt-infected cells. The western blots are representative of two independent experiments run in duplicate or triplicate ( K ). The titration ( A and D ), qRT-PCR ( B, D, H and J ), and ELISA ( I ) were done in biological triplicate and are representative of two independent experiments. The data analysis was done using a two-way ANOVA ( A, B, D, E, H, and J ) or one-way ANOVA ( I ) with Bonferroni’s or Tukey’s post-test for comparison between groups, respectively. P-value: *

    Techniques Used: Infection, Titration, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Fluorescence, Microscopy, Mutagenesis, Activation Assay, Western Blot, Quantitative RT-PCR

    39) Product Images from "Distinctive chaperonopathy in skeletal muscle associated with the dominant variant in DNAJB4"

    Article Title: Distinctive chaperonopathy in skeletal muscle associated with the dominant variant in DNAJB4

    Journal: bioRxiv

    doi: 10.1101/2022.07.26.501446

    Protein accumulation in the soleus muscles from Dnajb4 F90L/+ and Dnajb4 -/- mice at age of 28 months. a - t Modified Gomori trichrome (mGt) and immunofluorescent images staining for the indicated antibodies of the soleus muscles of Dnajb4 +/+ (WT) ( a , d ), Dnajb4 F90L/+ (F90L/+) ( b , e , g - n ) and Dnajb4 -/- (-/-) ( c , f , o - t ) mice at age of 28 months. j , n are magnified images of the white boxes of i , m , respectively. Muscle membranes are stained by anti-caveolin-3 (CAV3) antibodies (blue). Scale bar = 20 μm ( a-t ). u Immunoblot of lysates from the soleus muscles of Dnajb4 +/+ , Dnajb4 F90L/+ , and Dnajb4 -/- mice at 28 months of age. The bands were detected with antibodies against indicated proteins. GAPDH was used as a loading control. v Schematic diagram of the RIPA soluble/insoluble fractionation protocol. Gastrocnemius muscle lysates from Dnajb4 +/+ , Dnajb4 F90L/F90L (F90L/F90L), and Dnajb4 -/- mice at 13 months of age are separated into a total, soluble, and insoluble fraction. w Each fraction was immunoblotted for DNAJB4 and desmin. DES, desmin; FLNC, filamin C; HSP70, heat shock protein 70
    Figure Legend Snippet: Protein accumulation in the soleus muscles from Dnajb4 F90L/+ and Dnajb4 -/- mice at age of 28 months. a - t Modified Gomori trichrome (mGt) and immunofluorescent images staining for the indicated antibodies of the soleus muscles of Dnajb4 +/+ (WT) ( a , d ), Dnajb4 F90L/+ (F90L/+) ( b , e , g - n ) and Dnajb4 -/- (-/-) ( c , f , o - t ) mice at age of 28 months. j , n are magnified images of the white boxes of i , m , respectively. Muscle membranes are stained by anti-caveolin-3 (CAV3) antibodies (blue). Scale bar = 20 μm ( a-t ). u Immunoblot of lysates from the soleus muscles of Dnajb4 +/+ , Dnajb4 F90L/+ , and Dnajb4 -/- mice at 28 months of age. The bands were detected with antibodies against indicated proteins. GAPDH was used as a loading control. v Schematic diagram of the RIPA soluble/insoluble fractionation protocol. Gastrocnemius muscle lysates from Dnajb4 +/+ , Dnajb4 F90L/F90L (F90L/F90L), and Dnajb4 -/- mice at 13 months of age are separated into a total, soluble, and insoluble fraction. w Each fraction was immunoblotted for DNAJB4 and desmin. DES, desmin; FLNC, filamin C; HSP70, heat shock protein 70

    Techniques Used: Mouse Assay, Modification, Staining, Fractionation

    40) Product Images from "Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1) *"

    Article Title: Glycosylation at Asn211 Regulates the Activation State of the Discoidin Domain Receptor 1 (DDR1) *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.541102

    Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).
    Figure Legend Snippet: Sustained activation of N211Q DDR1b. COS1 cells expressing WT or N211Q DDR1b were serum-starved (18 h) before stimulation (2 h) with (+) 10 μg/ml rat tail collagen I ( Col. I ) or vehicle control (−), as described under “Experimental Procedures.” After stimulation, the media were aspirated, and the cells were washed thoroughly with warm PBS. The dishes were then supplemented with serum-free media and incubated at 37 °C for the indicated times. The cells were lysed with RIPA buffer, and the lysates were analyzed for receptor activation ( A ) and total receptor expression ( B ), as described in Fig. 2 . Black arrow in A indicates phosphorylated DDR1b, and white arrow in B indicates total DDR1b. Anti-Tyr(P) (α- pTyr ).

    Techniques Used: Activation Assay, Expressing, Incubation

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    Roche ripa buffer
    Ebola GP interacts with tetherin. ( A and B ) 293T cells were <t>transfected</t> with the indicated plasmids, and <t>RIPA</t> lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input
    Ripa Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche radioimmunoprecipitation assay buffer
    NiV M can bud from transfected cells and form virus-like particles. (a) HEK 293T cells were transfected as indicated with an expression plasmid encoding GFP (2 μg), HA NiV M (2 μg), NiV G (0.5 μg), or NiV F (1 μg). At 48 hours posttransfection, the culture medium was harvested and centrifuged through a 20% sucrose cushion to pellet VLPs, and the transfected cells were lysed in <t>radioimmunoprecipitation</t> assay buffer. Western blotting (IB) of lysates and the purified culture medium was performed with anti-GFP and anti-HA antibodies. (b) Purified HA NiV M VLPs were treated with TPCK-trypsin in the presence or absence of 1% Triton X-100 for 1 h at 37°C. They were then visualized by Western blotting with an anti-HA antibody. (c and d) Representative transmission electron micrographs of NiV VLPs produced by transfection of M alone (c) or M, G, and F (d), stained with 1% phosphotungstic acid, pH 7.0. Arrowheads indicate the studded appearance of NiV glycoproteins, G and/or F, at the VLP surface.
    Radioimmunoprecipitation Assay Buffer, supplied by Roche, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Ebola GP interacts with tetherin. ( A and B ) 293T cells were transfected with the indicated plasmids, and RIPA lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input

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

    Article Title: Tetherin-mediated restriction of filovirus budding is antagonized by the Ebola glycoprotein

    doi: 10.1073/pnas.0811014106

    Figure Lengend Snippet: Ebola GP interacts with tetherin. ( A and B ) 293T cells were transfected with the indicated plasmids, and RIPA lysates were harvested at 24 h. Proteins were immunoprecipitated with anti-AU1 ( A ) or GP antisera ( B ) and Western blotted as indicated. The input

    Article Snippet: Transfected 293T cells were lysed in RIPA buffer [50 mM Tris·HCl, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, protease inhibitors (Roche), pH 7.4] and sonicated.

    Techniques: Transfection, Immunoprecipitation, Western Blot

    An intact SMYD3 MYND domain is required for association with N-CoR and for transcriptional repression. (A) N-CoR co-immunoprecipitates with wildtype SMYD3 but not with SMYD3 MYND domain point mutant C49/S. 293T cells were co-transfected with N-CoR, N-terminal myc-tagged SMYD3 constructs indicated, and with empty vector (vector). 48 hours post-transfection, whole cell RIPA lysates (WCL) were prepared. Fractions of the lysates were subjected to anti-N-CoR co-immunoprecipitation and the remaining 50% served as input. Western analysis was performed with anti-myc antibodies. Myc-SMYD1B, previously shown to interact with N-CoR served as a positive control. (B) Schematic of GAL4-DNA binding domain (DBD) and GAL4-fusion constructs for wild type (GAL4-SMYD3) and MYND domain-mutated (GAL4-SMYD3-C49/S) two hybrid transcription assays. X denotes the location of the C49/S mutation. (C) GAL4-SMYD3 but not GAL4-SMYD3-C49/S represses transcription of a GAL4-UAS containing luciferase reporter. 293T cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 µg) together with GAL4-DBD, or with 1 or 2 µg (indicated as 1X or 2X) of GAL4-SMYD3 (black bars) or GAL4-SMYD3-C49/S (red bars). Transfection efficiencies were normalized to co-transfected renilla luciferase, and percent GAL4 activity was determined in relation to GAL4-DBD set at 100%.

    Journal: PLoS ONE

    Article Title: Structural and Functional Profiling of the Human Histone Methyltransferase SMYD3

    doi: 10.1371/journal.pone.0022290

    Figure Lengend Snippet: An intact SMYD3 MYND domain is required for association with N-CoR and for transcriptional repression. (A) N-CoR co-immunoprecipitates with wildtype SMYD3 but not with SMYD3 MYND domain point mutant C49/S. 293T cells were co-transfected with N-CoR, N-terminal myc-tagged SMYD3 constructs indicated, and with empty vector (vector). 48 hours post-transfection, whole cell RIPA lysates (WCL) were prepared. Fractions of the lysates were subjected to anti-N-CoR co-immunoprecipitation and the remaining 50% served as input. Western analysis was performed with anti-myc antibodies. Myc-SMYD1B, previously shown to interact with N-CoR served as a positive control. (B) Schematic of GAL4-DNA binding domain (DBD) and GAL4-fusion constructs for wild type (GAL4-SMYD3) and MYND domain-mutated (GAL4-SMYD3-C49/S) two hybrid transcription assays. X denotes the location of the C49/S mutation. (C) GAL4-SMYD3 but not GAL4-SMYD3-C49/S represses transcription of a GAL4-UAS containing luciferase reporter. 293T cells were transiently co-transfected with the 5XGAL4-SV40-luciferase reporter (1 µg) together with GAL4-DBD, or with 1 or 2 µg (indicated as 1X or 2X) of GAL4-SMYD3 (black bars) or GAL4-SMYD3-C49/S (red bars). Transfection efficiencies were normalized to co-transfected renilla luciferase, and percent GAL4 activity was determined in relation to GAL4-DBD set at 100%.

    Article Snippet: Immunoprecipitation (IP) and Western blotting 293T cells were transiently transfected, harvested 48 hours later, and then lysed in RIPA buffer (150 mM NaCl, 1% NP-40, 0.5% DOC, 50 mM Tris pH 8, 0.1% SDS) containing protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN).

    Techniques: Mutagenesis, Transfection, Construct, Plasmid Preparation, Immunoprecipitation, Western Blot, Positive Control, Binding Assay, Luciferase, Activity Assay

    Biochemical assessment of the avidity of CDV H-cSLAM interactions. (A to C) Coimmunoprecipitation assays. CDV H FLAG and HA-tagged wt cSLAM, mutant cSLAM, or lion SLAM were coexpressed in Vero cells and subsequently lysed with RIPA buffer 24 h posttransfection.

    Journal: Journal of Virology

    Article Title: Canine Distemper Virus Fusion Activation: Critical Role of Residue E123 of CD150/SLAM

    doi: 10.1128/JVI.02405-15

    Figure Lengend Snippet: Biochemical assessment of the avidity of CDV H-cSLAM interactions. (A to C) Coimmunoprecipitation assays. CDV H FLAG and HA-tagged wt cSLAM, mutant cSLAM, or lion SLAM were coexpressed in Vero cells and subsequently lysed with RIPA buffer 24 h posttransfection.

    Article Snippet: Vero cells in a six-well-plate format were transfected with 2-μg amounts of canine SLAM-expressing plasmids (wt or derived mutants) and 2 μg of wt pCI-H. At 24 h posttransfection, cells were lysed in RIPA buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS) containing protease inhibitor (cOmplete mix; Roche).

    Techniques: Mutagenesis

    NiV M can bud from transfected cells and form virus-like particles. (a) HEK 293T cells were transfected as indicated with an expression plasmid encoding GFP (2 μg), HA NiV M (2 μg), NiV G (0.5 μg), or NiV F (1 μg). At 48 hours posttransfection, the culture medium was harvested and centrifuged through a 20% sucrose cushion to pellet VLPs, and the transfected cells were lysed in radioimmunoprecipitation assay buffer. Western blotting (IB) of lysates and the purified culture medium was performed with anti-GFP and anti-HA antibodies. (b) Purified HA NiV M VLPs were treated with TPCK-trypsin in the presence or absence of 1% Triton X-100 for 1 h at 37°C. They were then visualized by Western blotting with an anti-HA antibody. (c and d) Representative transmission electron micrographs of NiV VLPs produced by transfection of M alone (c) or M, G, and F (d), stained with 1% phosphotungstic acid, pH 7.0. Arrowheads indicate the studded appearance of NiV glycoproteins, G and/or F, at the VLP surface.

    Journal: Journal of Virology

    Article Title: Mutation of YMYL in the Nipah Virus Matrix Protein Abrogates Budding and Alters Subcellular Localization ▿

    doi: 10.1128/JVI.01743-06

    Figure Lengend Snippet: NiV M can bud from transfected cells and form virus-like particles. (a) HEK 293T cells were transfected as indicated with an expression plasmid encoding GFP (2 μg), HA NiV M (2 μg), NiV G (0.5 μg), or NiV F (1 μg). At 48 hours posttransfection, the culture medium was harvested and centrifuged through a 20% sucrose cushion to pellet VLPs, and the transfected cells were lysed in radioimmunoprecipitation assay buffer. Western blotting (IB) of lysates and the purified culture medium was performed with anti-GFP and anti-HA antibodies. (b) Purified HA NiV M VLPs were treated with TPCK-trypsin in the presence or absence of 1% Triton X-100 for 1 h at 37°C. They were then visualized by Western blotting with an anti-HA antibody. (c and d) Representative transmission electron micrographs of NiV VLPs produced by transfection of M alone (c) or M, G, and F (d), stained with 1% phosphotungstic acid, pH 7.0. Arrowheads indicate the studded appearance of NiV glycoproteins, G and/or F, at the VLP surface.

    Article Snippet: Transfected cells were washed briefly with phosphate-buffered saline and lysed in radioimmunoprecipitation assay buffer (50 mM Tris [pH 7.4], 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% deoxycholate, 1% NP-40) supplemented with protease inhibitor cocktail (Complete; Roche, Mannheim, Germany).

    Techniques: Transfection, Expressing, Plasmid Preparation, Radio Immunoprecipitation, Western Blot, Purification, Transmission Assay, Produced, Staining