rnase inhibitor  (Millipore)


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    Name:
    Protector RNase Inhibitor
    Description:
    Protector RNase Inhibitor inactivates a wide spectrum of RNases including RNase A RNase B RNase T2Thus Protector RNase Inhibitor can help prevent RNase degradation in any application where RNases could caus problems For instance it can protect mRNA during cDNA synthesis reactions RT PCR in conventional thermal cyclers and qPCR systems or in vitro transcription translation reactions protect viral RNA during in vitro virus replication inhibit RNases during RNA isolation and purification be used in RNase protection assays help prepare RNase free antibodiesNote Protector RNase Inhibitor does not interfere with enzymes commonly used to prepare or analyze RNA
    Catalog Number:
    rnainh-ro
    Price:
    None
    Applications:
    Protector RNase Inhibitor is used to: . Protect mRNA in cDNA synthesis reactions, RT-PCR (in conventional thermal cyclers and qPCR systems), e.g., with the LightCycler(R) Instruments, in vitro transcription/translation system, RNase Protection Assay, in vitro RNA synthesis. Protect viral RNA during in vitro virus replication. Inhibit RNases during RNA isolation and purification. Help prepare RNase-free antibodies. Synthesize RNA probes for in situ hybridizationIt is useful in any application where RNases could be a potential problem.
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    Structured Review

    Millipore rnase inhibitor
    Protector RNase Inhibitor
    Protector RNase Inhibitor inactivates a wide spectrum of RNases including RNase A RNase B RNase T2Thus Protector RNase Inhibitor can help prevent RNase degradation in any application where RNases could caus problems For instance it can protect mRNA during cDNA synthesis reactions RT PCR in conventional thermal cyclers and qPCR systems or in vitro transcription translation reactions protect viral RNA during in vitro virus replication inhibit RNases during RNA isolation and purification be used in RNase protection assays help prepare RNase free antibodiesNote Protector RNase Inhibitor does not interfere with enzymes commonly used to prepare or analyze RNA
    https://www.bioz.com/result/rnase inhibitor/product/Millipore
    Average 97 stars, based on 110 article reviews
    Price from $9.99 to $1999.99
    rnase inhibitor - by Bioz Stars, 2020-08
    97/100 stars

    Images

    1) Product Images from "N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein"

    Article Title: N6-methyladenosine alters RNA structure to regulate binding of a low-complexity protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx141

    m 6 A alters RNA structure to recruit HNRNPG. ( A ) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. ( B ) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m 6 A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. ( C ) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m 6 A residue is indicated by a red dot. Ctrl, no nuclease added; V1; RNase V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. ( D ) Model showing that m 6 A disrupts RNA structure, exposes a motif that includes the m 6 A site, and recruits an RNA binding protein.
    Figure Legend Snippet: m 6 A alters RNA structure to recruit HNRNPG. ( A ) Sequence logo of the most enriched motif within HNRNPG PAR-CLIP peaks. ( B ) Left: secondary structure of the MALAT1 hairpin, showing the A-2,515-to-G/C/U mutations that were introduced at the m 6 A site. Right: quantification of relative HNRNPG pull-down with the original (2,515-A) and mutated (2,515-G/C/U) MALAT1 hairpins, normalized to pulled-down HIST1H1C. Data shown as mean; error bar = standard deviation; n = 3 biological replicates. ( C ) Left: structural probing of the unmethylated and methylated MALAT1 hairpins. The orange lines indicate regions with marked differences between the unmethylated and methylated hairpins. The location of the m 6 A residue is indicated by a red dot. Ctrl, no nuclease added; V1; RNase V1 digestion; S1, S1 nuclease digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. Right: secondary structure of the unmethylated and methylated MALAT1 hairpins, marked at their S1 nuclease (red lines) and V1 nuclease (green lines) cleavage sites. ( D ) Model showing that m 6 A disrupts RNA structure, exposes a motif that includes the m 6 A site, and recruits an RNA binding protein.

    Techniques Used: Sequencing, Cross-linking Immunoprecipitation, Standard Deviation, Methylation, RNA Binding Assay

    2) Product Images from "Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis"

    Article Title: Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-017-0003-8

    MID1 is connected to the mTOR-dependent translation initiation pathway a Identification of the MID1-interactome. MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation. MID1-binding proteins were identified by mass spectrometry. The mTOR-dependent translation initiation pathway is shown and the number of proteins identified belonging either to the eukaryotic translation initiation factor complex (eIF complex) or the ribosome are indicated. b Validation of the mass spectrometry results shown in a and Table 1 . MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation (IP FLAG). As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies to detect MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. c Effect of ribosome disassembly on the composition of the MID1-complex. MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of high concentrations of EDTA. Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, RPLP0, RPL5, RPS3. d To analyze the MID1-complex composition and its dependency on RNA, MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of RNAse. As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. e , f mTOR regulates translation of APP. e In vitro translation of in vitro transcribed APP-mRNA tagged to luciferase in the presence or absence of the mTOR-inhibitor temsirolimus. The level of translated luciferase reporter was measured in a luciferase assay. Columns represent mean values ± SEM. n = 3. * p
    Figure Legend Snippet: MID1 is connected to the mTOR-dependent translation initiation pathway a Identification of the MID1-interactome. MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation. MID1-binding proteins were identified by mass spectrometry. The mTOR-dependent translation initiation pathway is shown and the number of proteins identified belonging either to the eukaryotic translation initiation factor complex (eIF complex) or the ribosome are indicated. b Validation of the mass spectrometry results shown in a and Table 1 . MID1-FLAG was expressed in HEK293T cells and MID1-complexes were purified by immunoprecipitation (IP FLAG). As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies to detect MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. c Effect of ribosome disassembly on the composition of the MID1-complex. MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of high concentrations of EDTA. Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, RPLP0, RPL5, RPS3. d To analyze the MID1-complex composition and its dependency on RNA, MID1-FLAG was expressed in HEK293T cells and immunopurified (IP FLAG) in the presence or absence of RNAse. As negative control, unspecific IgG agarose beads were used (IgG). Immunoprecipitates were analyzed on western blots using specific antibodies for MID1-FLAG, eIF3A, eIF4G, RPLP0, RPL5, RPS3. e , f mTOR regulates translation of APP. e In vitro translation of in vitro transcribed APP-mRNA tagged to luciferase in the presence or absence of the mTOR-inhibitor temsirolimus. The level of translated luciferase reporter was measured in a luciferase assay. Columns represent mean values ± SEM. n = 3. * p

    Techniques Used: Purification, Immunoprecipitation, Binding Assay, Mass Spectrometry, Negative Control, Western Blot, In Vitro, Luciferase

    3) Product Images from "Efficient in vitro siRNA delivery and Intramuscular Gene Silencing using PEG-modified PAMAM Dendrimers"

    Article Title: Efficient in vitro siRNA delivery and Intramuscular Gene Silencing using PEG-modified PAMAM Dendrimers

    Journal: Molecular Pharmaceutics

    doi: 10.1021/mp3001364

    The protection effect of PEGylated-dendrimers on siRNA in the presence of RNase A. A: siRNA only (control); B: G5-PEG/siRNA dendriplexes formed at an N/P ratio of 10; C: G6-PEG/siRNA dendriplexes formed at an N/P ratio of 10. Note that both G5-PEG and
    Figure Legend Snippet: The protection effect of PEGylated-dendrimers on siRNA in the presence of RNase A. A: siRNA only (control); B: G5-PEG/siRNA dendriplexes formed at an N/P ratio of 10; C: G6-PEG/siRNA dendriplexes formed at an N/P ratio of 10. Note that both G5-PEG and

    Techniques Used:

    4) Product Images from "N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions"

    Article Title: N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions

    Journal: Nature

    doi: 10.1038/nature14234

    m 6 A is enriched in the vicinity of hnRNP C binding sites a, Schematic diagram of the CLIP-2dTLC protocol. IP, immunoprecipitation; nt, nucleotide. The RNase T1 used in our 2dTLC assay cleaves single-stranded RNA after guanosines, so the m 6 A/A ratio determined here represents the m 6 A fraction of all adenosines following guanosines. b , Analysis of crosslinked RNA-hnRNP C complexes (CLIP RNP) using denaturing gel electrophoresis (lanes 1 and 2). Positions of the protein size standards are shown on the left. hnRNP C IP RNA region (RNA samples within RNA-hnRNP C crosslinked complexes) were extracted from the gel slices marked by the red rectangle. c, Denaturing gel analyzing the size distribution for the hnRNP C PAR-CLIP RNA samples (lane 2). The RNA size standards were loaded in lanes 1 and 3.
    Figure Legend Snippet: m 6 A is enriched in the vicinity of hnRNP C binding sites a, Schematic diagram of the CLIP-2dTLC protocol. IP, immunoprecipitation; nt, nucleotide. The RNase T1 used in our 2dTLC assay cleaves single-stranded RNA after guanosines, so the m 6 A/A ratio determined here represents the m 6 A fraction of all adenosines following guanosines. b , Analysis of crosslinked RNA-hnRNP C complexes (CLIP RNP) using denaturing gel electrophoresis (lanes 1 and 2). Positions of the protein size standards are shown on the left. hnRNP C IP RNA region (RNA samples within RNA-hnRNP C crosslinked complexes) were extracted from the gel slices marked by the red rectangle. c, Denaturing gel analyzing the size distribution for the hnRNP C PAR-CLIP RNA samples (lane 2). The RNA size standards were loaded in lanes 1 and 3.

    Techniques Used: Binding Assay, Cross-linking Immunoprecipitation, Immunoprecipitation, Nucleic Acid Electrophoresis

    m 6 A increases the accessibility of U-tract to enhance hnRNP C binding a, Secondary structure of the MALAT1 hairpin with m 6 A methylation at 2,577 site shown in red 25 . Nucleotide position numbers correspond to their locations along the human MALAT1 transcript (NR_002819). b, RNA pull down showing hnRNP C preferably binds methylated RNA. c, The list of proteins with identified peptides by mass spectrometry in b . d, Recombinant hnRNP C1 binds stronger with MALAT1 2,577-m 6 A hairpin compared to the unmethylated hairpin, as determined by in vitro UV crosslinking assay 23 . e, hnRNP C shows binding around A2,577 site along MALAT1 in vivo , as determined by previously published hnRNP C iCLIP data 20 . The underlying genomic sequence is shown at the bottom with a red square marking the m 6 A2,577 site. The slight shift of the iCLIP signal to upstream of the U-tract binding site is likely due to the steric hindrance of the peptide fragment remaining on RNA which can cause reverse transcription to terminate more than one nucleotide upstream of the cross-link site 20 . f, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assay in Fig. 1e . To correct for sample loading difference, each band signal was normalized to the band signal of the immediate 3’ residue to the U-tract. n = 3, ± s.d., technical replicates. g, Quantitative of the RNase T1 cleavage signal from RNA structural mapping assay in Fig. 1e . Increased RNase T1 cleavage signal (single-stranded specific cleavage after guanosines) was observed due to the surrounding m 6 A residue. To correct for sample loading difference, the ratio for each band signal among all bands in each lane was calculated. The y-axis value Relative T1 cleavage = (m 6 A native /m 6 A denature )/(A native /A denature ). n = 2, technical replicates. h, Quantitative CMCT mapping showing increased signals for the U-tract bases around the U base-pairing with m 6 A. Quantitation of band signals within the U-tract region is shown on the right. n = 4, ± s.d., technical replicates.
    Figure Legend Snippet: m 6 A increases the accessibility of U-tract to enhance hnRNP C binding a, Secondary structure of the MALAT1 hairpin with m 6 A methylation at 2,577 site shown in red 25 . Nucleotide position numbers correspond to their locations along the human MALAT1 transcript (NR_002819). b, RNA pull down showing hnRNP C preferably binds methylated RNA. c, The list of proteins with identified peptides by mass spectrometry in b . d, Recombinant hnRNP C1 binds stronger with MALAT1 2,577-m 6 A hairpin compared to the unmethylated hairpin, as determined by in vitro UV crosslinking assay 23 . e, hnRNP C shows binding around A2,577 site along MALAT1 in vivo , as determined by previously published hnRNP C iCLIP data 20 . The underlying genomic sequence is shown at the bottom with a red square marking the m 6 A2,577 site. The slight shift of the iCLIP signal to upstream of the U-tract binding site is likely due to the steric hindrance of the peptide fragment remaining on RNA which can cause reverse transcription to terminate more than one nucleotide upstream of the cross-link site 20 . f, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assay in Fig. 1e . To correct for sample loading difference, each band signal was normalized to the band signal of the immediate 3’ residue to the U-tract. n = 3, ± s.d., technical replicates. g, Quantitative of the RNase T1 cleavage signal from RNA structural mapping assay in Fig. 1e . Increased RNase T1 cleavage signal (single-stranded specific cleavage after guanosines) was observed due to the surrounding m 6 A residue. To correct for sample loading difference, the ratio for each band signal among all bands in each lane was calculated. The y-axis value Relative T1 cleavage = (m 6 A native /m 6 A denature )/(A native /A denature ). n = 2, technical replicates. h, Quantitative CMCT mapping showing increased signals for the U-tract bases around the U base-pairing with m 6 A. Quantitation of band signals within the U-tract region is shown on the right. n = 4, ± s.d., technical replicates.

    Techniques Used: Binding Assay, Methylation, Mass Spectrometry, Recombinant, In Vitro, In Vivo, Sequencing, Mapping Assay, Quantitation Assay

    Increased accessibility of U-tracts enhances hnRNP C binding a, Structure probing of the 2,577A-to-U mutated MALAT1 hairpin (2,577-U), same annotation as in Fig. 1d . b, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assays as in a . To correct for sample loading difference, each band signal was normalized to the band signal of the 3’ most U of the U-tract. n = 2, technical replicates. c, Filter-binding curves displaying the binding affinities between recombinant hnRNP C1 and 2,577-U/A oligos. n = 3, ± s.d., technical replicates. d, Filter-binding results showing the binding affinities between recombinant hnRNP C1 and four mutated MALAT1 oligos. (i) Mutate G-C to C-C, A2,577: predicted to weaken the hairpin stem and increase hnRNP C binding. Results: binding improved from 722 nM K d to 142 nM (5-fold); (ii) Mutate G-C to C-C, m 6 A2,577: in this context of weaker stem, m 6 A is predicted to confer a smaller effect compared to wild-type hairpin. Result: improved binding only 2-fold instead of 8-fold; (iii) Restore C-C to C-G, A2,577: predicted to restore the hairpin stem and decrease hnRNP C binding compared to C-C mutant. Result: binding decreased by 6.4-fold; (iv) Restore C-C to C-G, m 6 A2,577: in this context of restored stem, m 6 A is again predicted to confer increased binding compared to A2,577 hairpin. Result: improved binding by 2.5-fold. n = 3 each, ± s.d., technical replicates. e, RNA alkaline hydrolysis terminal truncation assay showing recombinant hnRNP C1 binding to terminal truncated MALAT1 hairpin oligos (2,577 site m 6 A methylated or unmethylated). In this assay, 3′ radiolabeled MALAT1 2,577 hairpin oligos were terminal truncated by alkaline hydrolysis into RNA fragments which were then incubated with hnRNP C1 protein followed by filter binding wash steps. The remaining RNA on the filter paper was isolated and analyzed by denaturing gel electrophoresis, as indicated in the lane “C1-bound or C1-B”. “Input” refers to alkaline hydrolysis truncated RNA oligos used for incubation with hnRNP C1; “G-L or G-ladder” was generated from RNase T1 digestion; “Ctrl” refers to the intact MALAT1 hairpin without alkaline hydrolysis truncation. One pair of methylated/unmethylated truncated oligos (CUT1, marked by green arrows) was selected for subsequent biochemical analysis, due to their strong interaction with hnRNP C1. f, RNA terminal truncation assay as in e except 5′ 32 P-labeled oligos were used. One pair of methylated/unmethylated truncated oligos (CUT2, marked by green arrows) was selected for subsequent biochemical analysis. g , Structure probing of the CUT1 oligos using RNase V1 and nuclease S1 digestion, same annotation as in Fig. 1e . The red dot marks the m 6 A site and the red line marks the U-tract region. h, Structure probing of the CUT2 oligos using RNase V1 and nuclease S1 digestion, same annotation as in g . i, Truncated oligos with exposed U-tracts increased hnRNP C binding regardless of m 6 A. n = 3, ± s.d., technical replicates.
    Figure Legend Snippet: Increased accessibility of U-tracts enhances hnRNP C binding a, Structure probing of the 2,577A-to-U mutated MALAT1 hairpin (2,577-U), same annotation as in Fig. 1d . b, Quantification of the RNase V1 cleavage signal for the U-tract region from RNA structural mapping assays as in a . To correct for sample loading difference, each band signal was normalized to the band signal of the 3’ most U of the U-tract. n = 2, technical replicates. c, Filter-binding curves displaying the binding affinities between recombinant hnRNP C1 and 2,577-U/A oligos. n = 3, ± s.d., technical replicates. d, Filter-binding results showing the binding affinities between recombinant hnRNP C1 and four mutated MALAT1 oligos. (i) Mutate G-C to C-C, A2,577: predicted to weaken the hairpin stem and increase hnRNP C binding. Results: binding improved from 722 nM K d to 142 nM (5-fold); (ii) Mutate G-C to C-C, m 6 A2,577: in this context of weaker stem, m 6 A is predicted to confer a smaller effect compared to wild-type hairpin. Result: improved binding only 2-fold instead of 8-fold; (iii) Restore C-C to C-G, A2,577: predicted to restore the hairpin stem and decrease hnRNP C binding compared to C-C mutant. Result: binding decreased by 6.4-fold; (iv) Restore C-C to C-G, m 6 A2,577: in this context of restored stem, m 6 A is again predicted to confer increased binding compared to A2,577 hairpin. Result: improved binding by 2.5-fold. n = 3 each, ± s.d., technical replicates. e, RNA alkaline hydrolysis terminal truncation assay showing recombinant hnRNP C1 binding to terminal truncated MALAT1 hairpin oligos (2,577 site m 6 A methylated or unmethylated). In this assay, 3′ radiolabeled MALAT1 2,577 hairpin oligos were terminal truncated by alkaline hydrolysis into RNA fragments which were then incubated with hnRNP C1 protein followed by filter binding wash steps. The remaining RNA on the filter paper was isolated and analyzed by denaturing gel electrophoresis, as indicated in the lane “C1-bound or C1-B”. “Input” refers to alkaline hydrolysis truncated RNA oligos used for incubation with hnRNP C1; “G-L or G-ladder” was generated from RNase T1 digestion; “Ctrl” refers to the intact MALAT1 hairpin without alkaline hydrolysis truncation. One pair of methylated/unmethylated truncated oligos (CUT1, marked by green arrows) was selected for subsequent biochemical analysis, due to their strong interaction with hnRNP C1. f, RNA terminal truncation assay as in e except 5′ 32 P-labeled oligos were used. One pair of methylated/unmethylated truncated oligos (CUT2, marked by green arrows) was selected for subsequent biochemical analysis. g , Structure probing of the CUT1 oligos using RNase V1 and nuclease S1 digestion, same annotation as in Fig. 1e . The red dot marks the m 6 A site and the red line marks the U-tract region. h, Structure probing of the CUT2 oligos using RNase V1 and nuclease S1 digestion, same annotation as in g . i, Truncated oligos with exposed U-tracts increased hnRNP C binding regardless of m 6 A. n = 3, ± s.d., technical replicates.

    Techniques Used: Binding Assay, Recombinant, Mutagenesis, Methylation, Incubation, Isolation, Nucleic Acid Electrophoresis, Generated, Labeling

    m 6 A alters RNA structure to enhance hnRNP C binding a, m 6 A increases binding with nuclear proteins. b, RNA pull down showing hnRNP C preferably binds methylated RNA. n = 4, ± s.d., biological replicates. c, Filter binding showing m 6 A increases hnRNP C1 binding with respective apparent dissociation constant ( K d ) indicated at lower right; n = 3, ± s.d., technical replicates. d, RNA structural probing showing m 6 A disrupts local RNA structure highlighted in yellow. Ctrl, no nuclease added; V1, RNase V1 digestion; S1, nuclease S1 digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. The orange bars mark the structurally altered RNA regions in the presence of m 6 A (red dot). e, RNA pull down with mutated oligos. n = 3, ± s.d., technical replicates. f, Illustration of the m 6 A-switch model.
    Figure Legend Snippet: m 6 A alters RNA structure to enhance hnRNP C binding a, m 6 A increases binding with nuclear proteins. b, RNA pull down showing hnRNP C preferably binds methylated RNA. n = 4, ± s.d., biological replicates. c, Filter binding showing m 6 A increases hnRNP C1 binding with respective apparent dissociation constant ( K d ) indicated at lower right; n = 3, ± s.d., technical replicates. d, RNA structural probing showing m 6 A disrupts local RNA structure highlighted in yellow. Ctrl, no nuclease added; V1, RNase V1 digestion; S1, nuclease S1 digestion; T1, RNase T1 digestion; G-L, G-ladder; AH, alkaline hydrolysis. The orange bars mark the structurally altered RNA regions in the presence of m 6 A (red dot). e, RNA pull down with mutated oligos. n = 3, ± s.d., technical replicates. f, Illustration of the m 6 A-switch model.

    Techniques Used: Binding Assay, Methylation

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    Blocking Assay:

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    Incubation:

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    other:

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    Protease Inhibitor:

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    Lysis:

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    Immunofluorescence:

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    Millipore sb203580
    Adenovirus vector-induced expression of IP-10 involves ERK and p38 in vivo. (A) RNase protection assay for IP-10 and MIP-2 mRNA in DBA/2 mouse liver 1 h following the intravenous administration of 10 11 particles of AdCMVβgal. Mice were pretreated with <t>SB203580</t> ( n = 3), U0126 ( n = 3), or vehicle ( n = 3). SB203580 and U0126 blocked the induction of IP-10 mRNA by AdCMVβgal. In contrast, adenovirus-induced MIP-2 expression was unaffected by these compounds. (B) RNase protection assay for IP-10 mRNA 6 and 24 h following the intravenous administration of 10 11 particles of AdCMVβgal. Pretreatment with SB203580 did not affect vector-induced gene expression at these time points. GAPDH is used as a loading control. (C) Liver β-galactosidase activity and serum transaminase levels in AdCMVβgal-transduced mice (2.5 × 10 11 particles/mouse) treated with SB203580. β-Galactosidase activities were similar in SB203580- and vehicle-treated mice ( n = 3). Serum AST (white bars) and ALT (black bars) levels in AdCMVβgal-transduced mice at 24 h were unaffected by treatment with SB203580.
    Sb203580, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1013 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore gtp rac1 pull down assay
    CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of <t>GTP</t> (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of <t>Rac1</t> GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value
    Gtp Rac1 Pull Down Assay, supplied by Millipore, 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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    99
    Millipore rac1
    Targeting the NADPH oxidase through <t>Rac1</t> or p47phox influences HO-1 expression
    Rac1, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rac1/product/Millipore
    Average 99 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    rac1 - by Bioz Stars, 2020-08
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    Image Search Results


    Adenovirus vector-induced expression of IP-10 involves ERK and p38 in vivo. (A) RNase protection assay for IP-10 and MIP-2 mRNA in DBA/2 mouse liver 1 h following the intravenous administration of 10 11 particles of AdCMVβgal. Mice were pretreated with SB203580 ( n = 3), U0126 ( n = 3), or vehicle ( n = 3). SB203580 and U0126 blocked the induction of IP-10 mRNA by AdCMVβgal. In contrast, adenovirus-induced MIP-2 expression was unaffected by these compounds. (B) RNase protection assay for IP-10 mRNA 6 and 24 h following the intravenous administration of 10 11 particles of AdCMVβgal. Pretreatment with SB203580 did not affect vector-induced gene expression at these time points. GAPDH is used as a loading control. (C) Liver β-galactosidase activity and serum transaminase levels in AdCMVβgal-transduced mice (2.5 × 10 11 particles/mouse) treated with SB203580. β-Galactosidase activities were similar in SB203580- and vehicle-treated mice ( n = 3). Serum AST (white bars) and ALT (black bars) levels in AdCMVβgal-transduced mice at 24 h were unaffected by treatment with SB203580.

    Journal: Journal of Virology

    Article Title: Activation of p38 and ERK Signaling during Adenovirus Vector Cell Entry Lead to Expression of the C-X-C Chemokine IP-10

    doi: 10.1128/JVI.76.4.1559-1568.2002

    Figure Lengend Snippet: Adenovirus vector-induced expression of IP-10 involves ERK and p38 in vivo. (A) RNase protection assay for IP-10 and MIP-2 mRNA in DBA/2 mouse liver 1 h following the intravenous administration of 10 11 particles of AdCMVβgal. Mice were pretreated with SB203580 ( n = 3), U0126 ( n = 3), or vehicle ( n = 3). SB203580 and U0126 blocked the induction of IP-10 mRNA by AdCMVβgal. In contrast, adenovirus-induced MIP-2 expression was unaffected by these compounds. (B) RNase protection assay for IP-10 mRNA 6 and 24 h following the intravenous administration of 10 11 particles of AdCMVβgal. Pretreatment with SB203580 did not affect vector-induced gene expression at these time points. GAPDH is used as a loading control. (C) Liver β-galactosidase activity and serum transaminase levels in AdCMVβgal-transduced mice (2.5 × 10 11 particles/mouse) treated with SB203580. β-Galactosidase activities were similar in SB203580- and vehicle-treated mice ( n = 3). Serum AST (white bars) and ALT (black bars) levels in AdCMVβgal-transduced mice at 24 h were unaffected by treatment with SB203580.

    Article Snippet: Cell viability following treatment with SB203580 and U0126 as measured by trypan blue staining was > 90% and similar to that of untreated cells.

    Techniques: Plasmid Preparation, Expressing, In Vivo, Rnase Protection Assay, Mouse Assay, Activity Assay, AST Assay

    ERK and p38 activation by adenovirus vectors. (A) Immunoblotting for phosphorylated and total p44/42 ERK and p38 kinase in REC cells transduced with AdCMVβgal. ERK and p38 pathways were activated within 10 min and activation persisted for up to 3 h following transduction with AdCMVβgal. (B) RNase protection assay for IP-10 mRNA. REC cells were transduced with AdCMVβgal in the presence of the specific p38 kinase inhibitor SB203580 (30 μM), the MEK1-MEK2 inhibitor U0126 (10 μM), or both. SB203580 and U0126 inhibited AdCMVβgal-induced expression of IP-10 mRNA at 6 h. GAPDH is used as a loading control.

    Journal: Journal of Virology

    Article Title: Activation of p38 and ERK Signaling during Adenovirus Vector Cell Entry Lead to Expression of the C-X-C Chemokine IP-10

    doi: 10.1128/JVI.76.4.1559-1568.2002

    Figure Lengend Snippet: ERK and p38 activation by adenovirus vectors. (A) Immunoblotting for phosphorylated and total p44/42 ERK and p38 kinase in REC cells transduced with AdCMVβgal. ERK and p38 pathways were activated within 10 min and activation persisted for up to 3 h following transduction with AdCMVβgal. (B) RNase protection assay for IP-10 mRNA. REC cells were transduced with AdCMVβgal in the presence of the specific p38 kinase inhibitor SB203580 (30 μM), the MEK1-MEK2 inhibitor U0126 (10 μM), or both. SB203580 and U0126 inhibited AdCMVβgal-induced expression of IP-10 mRNA at 6 h. GAPDH is used as a loading control.

    Article Snippet: Cell viability following treatment with SB203580 and U0126 as measured by trypan blue staining was > 90% and similar to that of untreated cells.

    Techniques: Activation Assay, Transduction, Rnase Protection Assay, Expressing

    CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of GTP (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of Rac1 GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value

    Journal: Oncotarget

    Article Title: CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma

    doi: 10.18632/oncotarget.8638

    Figure Lengend Snippet: CRKL mediates ALK signaling and regulates cytoskeleton, cell migration and survival A. CRKL siRNA knockdown in H2228 and H3122. Protein lysates were extracted from cells treated with four individual CRLK siRNAs (20 nM) or their smartpool for 72 h, and subjected to Western blot analyses. The graph shows the quantification of CRKL proteins. B. Inhibition of Ras GTPase activity by CRKL siRNA knockdown. Upper left panel: Lysates of untreated H3122 cells were incubated with buffer (Ctrl), non-hydrolyzable analog of GTP (GTPγS as a positive control) or GDP (as a negative control). Upper right panel: Lysates of H3122 cells treated with or without CRKL siRNA were incubated with GTPγS. Active GTP-bound Ras was pulled down by GST-fusion Ras-binding domain of Raf1 (GST-Raf1-RBD) and detected by immunoblotting with Ras antibody. Lower panels: Total Ras is shown as the input control. C. Inhibition of Rac1 GTPase by CRKL siRNA knockdown. Upper left panel: H3122 cell lysates were incubated with buffer (Ctrl), or as positive and negative controls, with GTPγS and GDP, respectively. Upper right panel: H3122 cell lysates with or without CRKL siRNA knockdown were incubated with GTPγS. Active GTP-bound Rac1 was pulled down by GST-fusion p21 binding domain of p21-activated kinase 1 (Pak1) (GST-Pak1-PBD) and detected by immunoblotting with Rac1 antibody. Lower panels: Total Rac1 serves as the input control. D. Cell viability assessed 72h after treatment with or without CRKL siRNAs by MTS assay, E. Cell migration, assessed using quantitative Boyden Chamber technique (16h after plating), and F. Colony formation assays (10 days after plating) were also performed in H3122 and H2228 cells with/without CRKL siRNA knockdown. Data represent the means of at least three independent experiments and are presented as percentage of untreated cells (Student's t -test: p -value

    Article Snippet: GTP-RAS and GTP-RAC1 pull-down assay The pull-down of GTP-bound RAS and RAC1 was performed by the use of active RAS and RAC1 pull-down and detection kits (Millipore) respectively according to the manufacturer's instructions.

    Techniques: Migration, Western Blot, Inhibition, Activity Assay, Incubation, Positive Control, Negative Control, Binding Assay, MTS Assay

    Targeting the NADPH oxidase through Rac1 or p47phox influences HO-1 expression

    Journal: Cancer

    Article Title: Inhibition of the NADPH oxidase regulates HO-1 expression in chronic myeloid leukemia

    doi: 10.1002/cncr.26621

    Figure Lengend Snippet: Targeting the NADPH oxidase through Rac1 or p47phox influences HO-1 expression

    Article Snippet: We also blocked Rac1 activity by two additional approaches: transfection of a dominant negative mutant of Rac1, N.17-myc ( ) and by treating cells with a chemical inhibitor of Rac1, NSC23766 ( ).

    Techniques: Expressing

    NOX is required for the suppression of TGF-β1-induced growth inhibition by LTB 4 /BLT1 axis A. MCF10A cells infected with mock or shNOX4 lentiviruses were incubated with Et-OH (vehicle) or 100 nM of LTB 4 for 30 min and then stimulated with TGF-β1 at the indicated concentrations for 24 h. B. Stable Mv1Lu-pcDNA3 and Mv1Lu-BLT1 cell lines were pretreated with DMSO or 2.5 μM of DPI for 30 min and then stimulated with TGF-β1 at the indicated concentrations for 24 h. Immunoblotting data in the panel show the expression of BLT1. C. Mv1Lu cells infected with mock, shNOX4, or both shNOX4 and Smad3 EPSM lentiviruses were stimulated with TGF-β1 at the indicated concentrations for 24 h. Immunoblotting data in the panel show the expression of NOX4. Effect of TGF-β1 on cell proliferation was examined using the [ 3 H]thymidine incorporation assay. Data are the average of triplicates of three independent experiments and are expressed as percentage of growth (thymidine incorporation relative to control experiment). D. MCF10A and Mv1Lu cell lines that stably express pcDNA3 or BLT1 were treated with DMSO, 5 μM of Rac1 inhibitor, or 20 μM of apocyanin for 30 min and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Whole cell lysates were analyzed by immunoblot with a specific antibody against p27 KIP1 and p15 INK4B , respectively. β-actin levels were monitored as a control. E. HepG2 cells were cotransfected with p15 INK4B -luciferase reporter plasmid and either pcDNA3 or BLT1 plasmid together with control (scrambled, Scr) or NOX4 siRNAs, or Rac1N17 plasmid and then stimulated with 5 ng/ml of TGF-β1 for 24 h. F. HepG2 cells were cotransfected with p15 INK4B -luciferase reporter plasmid and either pcDNA3 or Smad3 EPSM plasmid together with pCMV, NOX4, or Rac1V12 plasmid and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Luciferase activities were normalized on the basis of β-galactosidase expression to adjust for variation in transfection efficiency. All quantitative data are shown as the mean ± SD of three independent experiments. ** p

    Journal: Oncotarget

    Article Title: The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region

    doi:

    Figure Lengend Snippet: NOX is required for the suppression of TGF-β1-induced growth inhibition by LTB 4 /BLT1 axis A. MCF10A cells infected with mock or shNOX4 lentiviruses were incubated with Et-OH (vehicle) or 100 nM of LTB 4 for 30 min and then stimulated with TGF-β1 at the indicated concentrations for 24 h. B. Stable Mv1Lu-pcDNA3 and Mv1Lu-BLT1 cell lines were pretreated with DMSO or 2.5 μM of DPI for 30 min and then stimulated with TGF-β1 at the indicated concentrations for 24 h. Immunoblotting data in the panel show the expression of BLT1. C. Mv1Lu cells infected with mock, shNOX4, or both shNOX4 and Smad3 EPSM lentiviruses were stimulated with TGF-β1 at the indicated concentrations for 24 h. Immunoblotting data in the panel show the expression of NOX4. Effect of TGF-β1 on cell proliferation was examined using the [ 3 H]thymidine incorporation assay. Data are the average of triplicates of three independent experiments and are expressed as percentage of growth (thymidine incorporation relative to control experiment). D. MCF10A and Mv1Lu cell lines that stably express pcDNA3 or BLT1 were treated with DMSO, 5 μM of Rac1 inhibitor, or 20 μM of apocyanin for 30 min and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Whole cell lysates were analyzed by immunoblot with a specific antibody against p27 KIP1 and p15 INK4B , respectively. β-actin levels were monitored as a control. E. HepG2 cells were cotransfected with p15 INK4B -luciferase reporter plasmid and either pcDNA3 or BLT1 plasmid together with control (scrambled, Scr) or NOX4 siRNAs, or Rac1N17 plasmid and then stimulated with 5 ng/ml of TGF-β1 for 24 h. F. HepG2 cells were cotransfected with p15 INK4B -luciferase reporter plasmid and either pcDNA3 or Smad3 EPSM plasmid together with pCMV, NOX4, or Rac1V12 plasmid and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Luciferase activities were normalized on the basis of β-galactosidase expression to adjust for variation in transfection efficiency. All quantitative data are shown as the mean ± SD of three independent experiments. ** p

    Article Snippet: AG1478, LY294002, KN-92, Calphostin C, U0126, PD169316, SP600125, BI-D1870, BIX02189, DPI, apocyanin, and Rac1 were obtained from Calbiochem (La Jolla, CA).

    Techniques: Inhibition, Infection, Incubation, Expressing, Thymidine Incorporation Assay, Stable Transfection, Luciferase, Plasmid Preparation, Transfection

    NOX is required for LTB 4 /BLT1-mediated induction of Smad3 linker region phosphorylation and inhibition of TGF-β1-stimulated SBE-Luc reporter activity A. MCF10A cells were pretreated with DMSO, 5 μM of Rac1 inhibitor, or 2.5 μM of DPI for 30 min, and then stimulated with EtOH (vehicle) or 100 nM of LTB 4 for 30 min. B. Stable HepG2-pcDNA3 and HepG2-BLT1 cell lines were infected with pLOK1 vector or shNOX4 lentiviruses. C. MCF10A cells were infected with mock or NOX4 lentiviruse. Whole cell extracts were analyzed by immunoblot with a specific antibody against NOX4, phospho-Smad3 (Thr179), and phospho-Smad3 (Ser208), respectively. β-actin levels were monitored as a control. Asterisk represents a phospho-Smad2 (Thr220). D. HepG2 cells co-transfected with SBE-luciferase reporter plasmid together with control (scrambled, Scr) or NOX4 siRNAs were treated with EtOH (vehicle) or 100 nM of LTB 4 for 30 min and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Upper panel show the immunoblotting result of plasmid transfection. Lower panel show the measured luciferase activity. E. HepG2 cells were co-transfected with SBE-luciferase reporter plasmid and either pcDNA3 or BLT1 plasmid together with control (scrambled, Scr) or NOX4 siRNAs and then stimulated with 5 ng/ml of TGF-β1 for 24 h. F. HepG2 cells co-transfected with SBE-luciferase reporter plasmid and either pcDNA3 or Smad3 EPSM plasmid together with pCMV or NOX4 plasmid and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Upper panel show the immunoblotting result of siRNA and plasmid transfection. Lower panel show the measured luciferase activity. G. HepG2 cells co-transfected with SBE-luciferase reporter plasmid together with pcDNA3 or Smad3 EPSM plasmid were incubated with or without 100 μM of H 2 O 2 for 30 min and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Luciferase activities were normalized on the basis of β-galactosidase expression to adjust for variation in transfection efficiency. All quantitative data are shown as the mean ± SD of three independent experiments. ** p

    Journal: Oncotarget

    Article Title: The proinflammatory LTB4/BLT1 signal axis confers resistance to TGF-β1-induced growth inhibition by targeting Smad3 linker region

    doi:

    Figure Lengend Snippet: NOX is required for LTB 4 /BLT1-mediated induction of Smad3 linker region phosphorylation and inhibition of TGF-β1-stimulated SBE-Luc reporter activity A. MCF10A cells were pretreated with DMSO, 5 μM of Rac1 inhibitor, or 2.5 μM of DPI for 30 min, and then stimulated with EtOH (vehicle) or 100 nM of LTB 4 for 30 min. B. Stable HepG2-pcDNA3 and HepG2-BLT1 cell lines were infected with pLOK1 vector or shNOX4 lentiviruses. C. MCF10A cells were infected with mock or NOX4 lentiviruse. Whole cell extracts were analyzed by immunoblot with a specific antibody against NOX4, phospho-Smad3 (Thr179), and phospho-Smad3 (Ser208), respectively. β-actin levels were monitored as a control. Asterisk represents a phospho-Smad2 (Thr220). D. HepG2 cells co-transfected with SBE-luciferase reporter plasmid together with control (scrambled, Scr) or NOX4 siRNAs were treated with EtOH (vehicle) or 100 nM of LTB 4 for 30 min and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Upper panel show the immunoblotting result of plasmid transfection. Lower panel show the measured luciferase activity. E. HepG2 cells were co-transfected with SBE-luciferase reporter plasmid and either pcDNA3 or BLT1 plasmid together with control (scrambled, Scr) or NOX4 siRNAs and then stimulated with 5 ng/ml of TGF-β1 for 24 h. F. HepG2 cells co-transfected with SBE-luciferase reporter plasmid and either pcDNA3 or Smad3 EPSM plasmid together with pCMV or NOX4 plasmid and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Upper panel show the immunoblotting result of siRNA and plasmid transfection. Lower panel show the measured luciferase activity. G. HepG2 cells co-transfected with SBE-luciferase reporter plasmid together with pcDNA3 or Smad3 EPSM plasmid were incubated with or without 100 μM of H 2 O 2 for 30 min and then stimulated with 5 ng/ml of TGF-β1 for 24 h. Luciferase activities were normalized on the basis of β-galactosidase expression to adjust for variation in transfection efficiency. All quantitative data are shown as the mean ± SD of three independent experiments. ** p

    Article Snippet: AG1478, LY294002, KN-92, Calphostin C, U0126, PD169316, SP600125, BI-D1870, BIX02189, DPI, apocyanin, and Rac1 were obtained from Calbiochem (La Jolla, CA).

    Techniques: Inhibition, Activity Assay, Infection, Plasmid Preparation, Transfection, Luciferase, Incubation, Expressing