Structured Review

Santa Cruz Biotechnology rabbit polyclonal ab
Runx2 transcription factor targets Smads to subnuclear sites. HeLa cells, which lack endogenous Runx proteins, were transfected with 0.5 μg of expression construct for Flag Smad5 together with HA-Runx2 expression vector. BMP2-treated or untreated cells were processed for WC ( a – c , untreated cells; g – i , BMP2-treated cells) or NM-IF ( d – f , untreated cells; j – l , BMP2-treated cells) preparation and in situ immunofluorescence 24 h after transfection. Smad5 was detected with mouse mAb against Flag tag whereas Runx2 was detected by a rabbit <t>polyclonal</t> Ab against HA tag. To confirm that the activation of Smad5 by BMP2 is required for nuclear accumulation and consequent subnuclear targeting of Smad5, a dominant negative inhibitor of BMP signaling, BMPRI (KR), was expressed along with expression constructs for HA-Runx2 and Flag-Smad5. Cells were treated with BMP2 and were processed for WC ( m – o ) or NM-IF ( p – r ) preparation and in situ immunofluorescence as described.
Rabbit Polyclonal Ab, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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93/100 stars

Images

1) Product Images from "Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites"

Article Title: Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites

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

doi: 10.1073/pnas.112664499

Runx2 transcription factor targets Smads to subnuclear sites. HeLa cells, which lack endogenous Runx proteins, were transfected with 0.5 μg of expression construct for Flag Smad5 together with HA-Runx2 expression vector. BMP2-treated or untreated cells were processed for WC ( a – c , untreated cells; g – i , BMP2-treated cells) or NM-IF ( d – f , untreated cells; j – l , BMP2-treated cells) preparation and in situ immunofluorescence 24 h after transfection. Smad5 was detected with mouse mAb against Flag tag whereas Runx2 was detected by a rabbit polyclonal Ab against HA tag. To confirm that the activation of Smad5 by BMP2 is required for nuclear accumulation and consequent subnuclear targeting of Smad5, a dominant negative inhibitor of BMP signaling, BMPRI (KR), was expressed along with expression constructs for HA-Runx2 and Flag-Smad5. Cells were treated with BMP2 and were processed for WC ( m – o ) or NM-IF ( p – r ) preparation and in situ immunofluorescence as described.
Figure Legend Snippet: Runx2 transcription factor targets Smads to subnuclear sites. HeLa cells, which lack endogenous Runx proteins, were transfected with 0.5 μg of expression construct for Flag Smad5 together with HA-Runx2 expression vector. BMP2-treated or untreated cells were processed for WC ( a – c , untreated cells; g – i , BMP2-treated cells) or NM-IF ( d – f , untreated cells; j – l , BMP2-treated cells) preparation and in situ immunofluorescence 24 h after transfection. Smad5 was detected with mouse mAb against Flag tag whereas Runx2 was detected by a rabbit polyclonal Ab against HA tag. To confirm that the activation of Smad5 by BMP2 is required for nuclear accumulation and consequent subnuclear targeting of Smad5, a dominant negative inhibitor of BMP signaling, BMPRI (KR), was expressed along with expression constructs for HA-Runx2 and Flag-Smad5. Cells were treated with BMP2 and were processed for WC ( m – o ) or NM-IF ( p – r ) preparation and in situ immunofluorescence as described.

Techniques Used: Transfection, Expressing, Construct, Plasmid Preparation, In Situ, Immunofluorescence, FLAG-tag, Activation Assay, Dominant Negative Mutation, IF-P

Runx factors specify subnuclear localization of Smads. HeLa cells were transfected with 0.5 μg of expression construct for Flag Smad1 along with Runx1, Runx2, or Runx3 expression vectors. Cells were treated with BMP2, and Smad1 was examined for association with Runx factors in the nuclear matrix (NM-IF preparation) by in situ immunofluorescence 24 h after transfection. Smad1 was detected with mouse mAb against Flag tag. Runx proteins were detected with rabbit polyclonal Abs at a dilution of 1:200. Images were taken by a Zeiss Axioplan microscope coupled with a charge-coupled device (CCD) camera and were processed for deconvulation microscopy with METAMORPH bioimaging software.
Figure Legend Snippet: Runx factors specify subnuclear localization of Smads. HeLa cells were transfected with 0.5 μg of expression construct for Flag Smad1 along with Runx1, Runx2, or Runx3 expression vectors. Cells were treated with BMP2, and Smad1 was examined for association with Runx factors in the nuclear matrix (NM-IF preparation) by in situ immunofluorescence 24 h after transfection. Smad1 was detected with mouse mAb against Flag tag. Runx proteins were detected with rabbit polyclonal Abs at a dilution of 1:200. Images were taken by a Zeiss Axioplan microscope coupled with a charge-coupled device (CCD) camera and were processed for deconvulation microscopy with METAMORPH bioimaging software.

Techniques Used: Transfection, Expressing, Construct, In Situ, Immunofluorescence, FLAG-tag, Microscopy, Software

Runx2 is required for subnuclear targeting of Smad5. ( a ) A schematic of Runx2 protein. The region where Smads interact is shown. The Y428A mutation, which disrupts association of Runx2 with the nuclear matrix, was introduced by PCR-based site-directed mutagenesis. QA, poly glutamine-poly alanine stretch; RHD, runt homology domain; NMTS, nuclear matrix-targeting signal; VWRPY, a highly conserved motif that mediate interactions of Runx2 with groucho /TLE proteins. ( b ) ROS17/2.8 cells grown in 100-mm plates were transfected with 10 μg each of expression constructs for Flag-Smad1 and wild-type HA-Runx2 or HA-Runx2 Y428A mutant. Cells were treated with 300 ng/ml of BMP2 for 24 h and processed for immunoprecipitation. One microgram Ab against Flag tag was used for immunoprecipitation. The immunoprecipitated complex was resolved by 8% SDS/PAGE. Wild-type or mutant Runx2 proteins were detected with mouse mAb against HA tag (dilution 1:2000). HeLa cells were transfected with 0.5 μg of expression constructs for Flag-Smad1 along with HA-tagged wild-type or Runx2 Y428A mutant proteins. Cells were treated with BMP2 and subjected to WC ( c – e ) or NM-IF ( f – h ) preparation and double-labeled in situ immunofluorescence with mouse mAb against Flag tag and rabbit polyclonal Ab against HA tag.
Figure Legend Snippet: Runx2 is required for subnuclear targeting of Smad5. ( a ) A schematic of Runx2 protein. The region where Smads interact is shown. The Y428A mutation, which disrupts association of Runx2 with the nuclear matrix, was introduced by PCR-based site-directed mutagenesis. QA, poly glutamine-poly alanine stretch; RHD, runt homology domain; NMTS, nuclear matrix-targeting signal; VWRPY, a highly conserved motif that mediate interactions of Runx2 with groucho /TLE proteins. ( b ) ROS17/2.8 cells grown in 100-mm plates were transfected with 10 μg each of expression constructs for Flag-Smad1 and wild-type HA-Runx2 or HA-Runx2 Y428A mutant. Cells were treated with 300 ng/ml of BMP2 for 24 h and processed for immunoprecipitation. One microgram Ab against Flag tag was used for immunoprecipitation. The immunoprecipitated complex was resolved by 8% SDS/PAGE. Wild-type or mutant Runx2 proteins were detected with mouse mAb against HA tag (dilution 1:2000). HeLa cells were transfected with 0.5 μg of expression constructs for Flag-Smad1 along with HA-tagged wild-type or Runx2 Y428A mutant proteins. Cells were treated with BMP2 and subjected to WC ( c – e ) or NM-IF ( f – h ) preparation and double-labeled in situ immunofluorescence with mouse mAb against Flag tag and rabbit polyclonal Ab against HA tag.

Techniques Used: Mutagenesis, Polymerase Chain Reaction, Transfection, Expressing, Construct, Immunoprecipitation, FLAG-tag, SDS Page, Labeling, In Situ, Immunofluorescence

2) Product Images from "Epstein-Barr Virus Infection of Na?ve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology"

Article Title: Epstein-Barr Virus Infection of Na?ve B Cells In Vitro Frequently Selects Clones with Mutated Immunoglobulin Genotypes: Implications for Virus Biology

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1002697

CDR3 spectratype analysis of mitogen-activated B blasts and LCL cultures derived from naive B cells. (A) Results of capillary gel electrophoresis of IgH PCR amplification products from uninfected naïve B cells, B CD40L/IL4 stimulated naive B blasts and naive B cell-derived LCLs established from donor 11 (tested at 6 and 9 weeks). A bell-shaped curve of PCR product peak sizes seen in the uninfected control culture and in the naïve B blast cultures indicates a polyclonal B cell population, while the small number of distinct peaks visible in the LCL cultures indicate that the population is dominated by a small number of clones. (B) Results of capillary gel electrophoresis of IgH PCR amplification products from naive B cell-derived LCLs established from donor 8. While a broad distribution of CDR3 lengths is observed in the uninfected control culture and 4 week LCL, only a small number of distinct peaks are visible in the LCL cultures at later time points indicating that the population is evolving towards monoclonality.
Figure Legend Snippet: CDR3 spectratype analysis of mitogen-activated B blasts and LCL cultures derived from naive B cells. (A) Results of capillary gel electrophoresis of IgH PCR amplification products from uninfected naïve B cells, B CD40L/IL4 stimulated naive B blasts and naive B cell-derived LCLs established from donor 11 (tested at 6 and 9 weeks). A bell-shaped curve of PCR product peak sizes seen in the uninfected control culture and in the naïve B blast cultures indicates a polyclonal B cell population, while the small number of distinct peaks visible in the LCL cultures indicate that the population is dominated by a small number of clones. (B) Results of capillary gel electrophoresis of IgH PCR amplification products from naive B cell-derived LCLs established from donor 8. While a broad distribution of CDR3 lengths is observed in the uninfected control culture and 4 week LCL, only a small number of distinct peaks are visible in the LCL cultures at later time points indicating that the population is evolving towards monoclonality.

Techniques Used: Derivative Assay, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification

3) Product Images from "Differential Incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and Major Histocompatibility Complex Class I and II Molecules into Human Immunodeficiency Virus Type 1 Virions and Microvesicles: Implications for Viral Pathogenesis and Immune Regulation"

Article Title: Differential Incorporation of CD45, CD80 (B7-1), CD86 (B7-2), and Major Histocompatibility Complex Class I and II Molecules into Human Immunodeficiency Virus Type 1 Virions and Microvesicles: Implications for Viral Pathogenesis and Immune Regulation

Journal: Journal of Virology

doi: 10.1128/JVI.75.13.6173-6182.2001

Virion and microvesicle preparations contain high levels of CD45, CD55, CD86, MHC-I, and MHC-II but not CD80. Virions and microvesicles derived from the T1, T2, TBLCL-CD4, and H9 cell lines were purified by sucrose density gradient ultracentrifugation. TBLCL-CD4 cell lysates served as a positive control and a way to determine the sensitivities of the different antibodies in the Western blot assays. Virion and microvesicle preparations (50 μg of total protein per lane) and the TBLCL-CD4 lysates were analyzed on an SDS–5 to 20% nondenaturing polyacrylamide gel under reducing or nonreducing conditions. Immunoblots were probed with a MAb to CD45 (HI30), a polyclonal serum to CD55 (H-319), a goat polyclonal serum to CD80 (N-20), a MAb to CD86 (IT2.2), a MAb to MHC-I, and a MAb to MHC-II (L243). The results shown are representative of at least three independent Western blot assays for each protein.
Figure Legend Snippet: Virion and microvesicle preparations contain high levels of CD45, CD55, CD86, MHC-I, and MHC-II but not CD80. Virions and microvesicles derived from the T1, T2, TBLCL-CD4, and H9 cell lines were purified by sucrose density gradient ultracentrifugation. TBLCL-CD4 cell lysates served as a positive control and a way to determine the sensitivities of the different antibodies in the Western blot assays. Virion and microvesicle preparations (50 μg of total protein per lane) and the TBLCL-CD4 lysates were analyzed on an SDS–5 to 20% nondenaturing polyacrylamide gel under reducing or nonreducing conditions. Immunoblots were probed with a MAb to CD45 (HI30), a polyclonal serum to CD55 (H-319), a goat polyclonal serum to CD80 (N-20), a MAb to CD86 (IT2.2), a MAb to MHC-I, and a MAb to MHC-II (L243). The results shown are representative of at least three independent Western blot assays for each protein.

Techniques Used: Derivative Assay, Purification, Positive Control, Western Blot

Related Articles

other:

Article Title: Evidence for Dsg3 in regulating Src signaling by competing with it for binding to caveolin-1
Article Snippet: 2.1 Antibodies The mouse monoclonal and rabbit polyclonal Abs used were: 5H10, mouse Ab against Dsg3 (Santa Cruz Biotechnology, Inc); HECD-1, mouse anti-E-cadherin (Abcam); Src (32G6) rabbit mAb and phospho-Src family (Tyr416) (Cell Signaling); rabbit caveolin-1 Ab (Cell Signaling); mouse mAb against caveolin-1 (Santa Cruz Biotechnology); rabbit anti beta actin-loading control ab8227 (Abcam); Secondary Abs were Alexa Fluor 488 conjugated goat anti-mouse IgG and Alexa Fluor 546 conjugated goat anti-rabbit IgG (Invitrogen).

Article Title: Aurora-A kinase interacting protein 1 (AURKAIP1) promotes Aurora-A degradation through an alternative ubiquitin-independent pathway
Article Snippet: Mouse monoclonal anti-FLAG M2 antibody (diluted 1:2000; Stratagene); rabbit polyclonal anti-FLAG (diluted 1:2000 Sigma); mouse monoclonal anti-β tubulin antibody (diluted 1:1000; Sigma); mouse monoclonal anti-(HA tag) (diluted 1:2000; Sigma); mouse monoclonal anti-IAK1 (Aurora-A kinase) (diluted 1:1000; BD Transduction); rabbit polyclonal anti-(cyclin B1) antibody (diluted 1:3000; Santa Cruz Biotechnology); and mouse monoclonal anti-(His6 tag) antibody (diluted 1:1000; Sigma), were used.

Immunofluorescence:

Article Title: Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models
Article Snippet: .. Antibodies For immunofluorescence microscopy the following primary (1) and secondary (2) antibodies (Abs) were used: 1) rat monoclonal Abs to Ki67 (Dako, 1∶100) and to CD29 (9EG7, BD Pharmingen, 1∶100), goat polyclonal Ab to vimentin (C20, Santa Cruz, 1∶100), and rabbit polyclonal Ab to fibronectin (H-200, Santa Cruz, 1∶100); and 2) donkey anti-goat IgG Alexa Fluor 488 (Invitrogen), goat anti-rat IgG Alexa Fluor 488 (Invitrogen), and goat anti-rabbit IgG DyLightTM 649 (Jackson ImmunoResearch Laboratories, Inc.). .. Rhodamine Phalloidin (Life Technologies) was used in a dilution of 1∶200.

Microscopy:

Article Title: Multiplex Profiling of Cellular Invasion in 3D Cell Culture Models
Article Snippet: .. Antibodies For immunofluorescence microscopy the following primary (1) and secondary (2) antibodies (Abs) were used: 1) rat monoclonal Abs to Ki67 (Dako, 1∶100) and to CD29 (9EG7, BD Pharmingen, 1∶100), goat polyclonal Ab to vimentin (C20, Santa Cruz, 1∶100), and rabbit polyclonal Ab to fibronectin (H-200, Santa Cruz, 1∶100); and 2) donkey anti-goat IgG Alexa Fluor 488 (Invitrogen), goat anti-rat IgG Alexa Fluor 488 (Invitrogen), and goat anti-rabbit IgG DyLightTM 649 (Jackson ImmunoResearch Laboratories, Inc.). .. Rhodamine Phalloidin (Life Technologies) was used in a dilution of 1∶200.

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    Santa Cruz Biotechnology anti tlr4
    Expression levels of <t>TLR4,</t> NK-1, and Na + /K + -ATPase were studied using Western blot analysis. Astrocytes were cultivated in 5.5 mM glucose the whole cultivation period. The cells were incubated with LPS (10 ng/ml) for 24 h, when incubated with LPS for 24 h followed by LPS, 25 mM glucose, a combination of naloxone (Nal) (10 − 12 M), endomorphin-1 (EM-1) (10 − 6 M), and levetiracetam (Lev) (10 − 4 M), or a combination of sildenafil (Sild) (1 μM) and vitamin D3 (D3) (100 nM), or combination of all substances for another 24 h. Unstimulated cells were used as controls. The level of significance was calculated against LPS (5.5) and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P
    Anti Tlr4, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti tlr4/product/Santa Cruz Biotechnology
    Average 93 stars, based on 97 article reviews
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    92
    Santa Cruz Biotechnology rabbit polyclonal anti caspase 1 p10
    Activation of IL-1β induced by CSFV infection was dependent on caspase 1. (A) The caspase 1 was activated by CSFV infection. PBMCs were mock treated or infected with CSFV at a MOI of 1 or 0.1 for 6 h. (B) The expression of pro-caspase 1 and the cleavage of caspase-1p10 protein were identified by western blot analysis following CSFV infection (MOI of 1 or 0.1). (C) Following treatment with the caspase 1 inhibitor Ac-yVAD-CHO, the activation of IL-1β induced by CSFV was inhibited. (D) The expression of caspase 1 precursor and the subunit <t>p20/P10</t> proteins were not affected by treatment with AC-yVAD-CHO. (E) Caspase 1 activity was dose-dependently inhibited by AC-yVAD-CHO, and CSFV activated caspase 1 activity at an MOI of 1. * P
    Rabbit Polyclonal Anti Caspase 1 P10, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti caspase 1 p10/product/Santa Cruz Biotechnology
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    93
    Santa Cruz Biotechnology anti cd2ap antibody
    APOL1 risk alleles destabilize the adherens complex. ( A ) Protein blots of DPDs stably over-expressing Vector (V), G0, G1, and G2 were probed for APOL1, nephrin, <t>CD2AP,</t> dynamin, CTSL, and GAPDH (n = 5–7). Representative gels are displayed. ( B ) Protein blots of the above lysates were probed for dendrin and reprobed for GAPDH (n = 5). ( C – H ) Cumulative densitometric data on each variable (protein/GAPDH) are shown as dot plots. *
    Anti Cd2ap Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 1 article reviews
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    93
    Santa Cruz Biotechnology rabbit polyclonal katanin p60 antibody
    Western blotting results of Elk1 and Elk1-3R effects on both endogenous <t>katanin-p60</t> and spastin proteins. (A) Analysis of SH-SY5Y transfection efficacy with His-tag including pCMV6_Elk1 and pCMV6_Elk1-3R vectors. (B) Western blotting results showing katanin-p60 level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (C) Quantification of katanin-p60 level was performed by normalizing band intensities of katanin-p60 to band intensities of ß-actin. Results indicated that both Elk1 and Elk1-3R decreased the katanin-p60 level significantly and the difference between them was not significant. SEM values are 0, 0.01, and 0.02, respectively (n = 4). (D) Western blotting results showing spastin level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (E) Quantification of spastin level was done as previously described. Results indicated that there was no significant spastin level difference between Elk1 and Elk1-3R transfected cells. However, they were significant compared to untransfected cells. SEM values are 0, 0.03, and 0.03, respectively (n = 4).
    Rabbit Polyclonal Katanin P60 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Expression levels of TLR4, NK-1, and Na + /K + -ATPase were studied using Western blot analysis. Astrocytes were cultivated in 5.5 mM glucose the whole cultivation period. The cells were incubated with LPS (10 ng/ml) for 24 h, when incubated with LPS for 24 h followed by LPS, 25 mM glucose, a combination of naloxone (Nal) (10 − 12 M), endomorphin-1 (EM-1) (10 − 6 M), and levetiracetam (Lev) (10 − 4 M), or a combination of sildenafil (Sild) (1 μM) and vitamin D3 (D3) (100 nM), or combination of all substances for another 24 h. Unstimulated cells were used as controls. The level of significance was calculated against LPS (5.5) and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P

    Journal: Journal of Neuroinflammation

    Article Title: Anti-inflammatory effects induced by pharmaceutical substances on inflammatory active brain astrocytes—promising treatment of neuroinflammation

    doi: 10.1186/s12974-018-1361-8

    Figure Lengend Snippet: Expression levels of TLR4, NK-1, and Na + /K + -ATPase were studied using Western blot analysis. Astrocytes were cultivated in 5.5 mM glucose the whole cultivation period. The cells were incubated with LPS (10 ng/ml) for 24 h, when incubated with LPS for 24 h followed by LPS, 25 mM glucose, a combination of naloxone (Nal) (10 − 12 M), endomorphin-1 (EM-1) (10 − 6 M), and levetiracetam (Lev) (10 − 4 M), or a combination of sildenafil (Sild) (1 μM) and vitamin D3 (D3) (100 nM), or combination of all substances for another 24 h. Unstimulated cells were used as controls. The level of significance was calculated against LPS (5.5) and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P

    Article Snippet: The membranes were probed with anti-TLR4 (rabbit polyclonal, 1:500) (Santa Cruz Biotech Inc., Dallas, TX, USA), anti-NK-1 (rabbit polyclonal, 1:1000) (LifeSpan BioSciences Inc., Seattle, USA), anti-PAR-2 (rabbit polyclonal, Santa Cruz Biotech Inc), or a mouse monoclonal primary antibody against Na+ /K+ -ATPase (α-subunit) (Sigma Aldrich) diluted 1:250, washed four time for 2 min with TBST, followed with the secondary horseradish peroxidase (HRP)-conjugated antibodies, donkey anti-mouse, or anti-rabbit F(ab’)2 fragment (Jackson ImmunoResearch) diluted 1:10000, also washed several times in TBST.

    Techniques: Expressing, Western Blot, Incubation

    Expression levels of the receptors TLR4, NK-1, and PAR-2 were studied using Western blot analysis. Astrocytes were cultivated in 5.5 mM or 25 mM glucose the whole cultivation period. The astrocytes were incubated with LPS (10 ng/ml) for 24 h or incubated with LPS for 24 h followed by LPS and sildenafil (Sild) (1 μM), or combination of all substances for another 24 h; unstimulated cells were used as controls ©. Statistical analysis: the level of significance was calculated against LPS (5.5) and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P

    Journal: Journal of Neuroinflammation

    Article Title: Anti-inflammatory effects induced by pharmaceutical substances on inflammatory active brain astrocytes—promising treatment of neuroinflammation

    doi: 10.1186/s12974-018-1361-8

    Figure Lengend Snippet: Expression levels of the receptors TLR4, NK-1, and PAR-2 were studied using Western blot analysis. Astrocytes were cultivated in 5.5 mM or 25 mM glucose the whole cultivation period. The astrocytes were incubated with LPS (10 ng/ml) for 24 h or incubated with LPS for 24 h followed by LPS and sildenafil (Sild) (1 μM), or combination of all substances for another 24 h; unstimulated cells were used as controls ©. Statistical analysis: the level of significance was calculated against LPS (5.5) and analyzed using one-way ANOVA followed by Dunnett’s multiple comparisons test. *P

    Article Snippet: The membranes were probed with anti-TLR4 (rabbit polyclonal, 1:500) (Santa Cruz Biotech Inc., Dallas, TX, USA), anti-NK-1 (rabbit polyclonal, 1:1000) (LifeSpan BioSciences Inc., Seattle, USA), anti-PAR-2 (rabbit polyclonal, Santa Cruz Biotech Inc), or a mouse monoclonal primary antibody against Na+ /K+ -ATPase (α-subunit) (Sigma Aldrich) diluted 1:250, washed four time for 2 min with TBST, followed with the secondary horseradish peroxidase (HRP)-conjugated antibodies, donkey anti-mouse, or anti-rabbit F(ab’)2 fragment (Jackson ImmunoResearch) diluted 1:10000, also washed several times in TBST.

    Techniques: Expressing, Western Blot, Incubation

    Activation of IL-1β induced by CSFV infection was dependent on caspase 1. (A) The caspase 1 was activated by CSFV infection. PBMCs were mock treated or infected with CSFV at a MOI of 1 or 0.1 for 6 h. (B) The expression of pro-caspase 1 and the cleavage of caspase-1p10 protein were identified by western blot analysis following CSFV infection (MOI of 1 or 0.1). (C) Following treatment with the caspase 1 inhibitor Ac-yVAD-CHO, the activation of IL-1β induced by CSFV was inhibited. (D) The expression of caspase 1 precursor and the subunit p20/P10 proteins were not affected by treatment with AC-yVAD-CHO. (E) Caspase 1 activity was dose-dependently inhibited by AC-yVAD-CHO, and CSFV activated caspase 1 activity at an MOI of 1. * P

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Activation of Interleukin-1β Release by the Classical Swine Fever Virus Is Dependent on the NLRP3 Inflammasome, Which Affects Virus Growth in Monocytes

    doi: 10.3389/fcimb.2018.00225

    Figure Lengend Snippet: Activation of IL-1β induced by CSFV infection was dependent on caspase 1. (A) The caspase 1 was activated by CSFV infection. PBMCs were mock treated or infected with CSFV at a MOI of 1 or 0.1 for 6 h. (B) The expression of pro-caspase 1 and the cleavage of caspase-1p10 protein were identified by western blot analysis following CSFV infection (MOI of 1 or 0.1). (C) Following treatment with the caspase 1 inhibitor Ac-yVAD-CHO, the activation of IL-1β induced by CSFV was inhibited. (D) The expression of caspase 1 precursor and the subunit p20/P10 proteins were not affected by treatment with AC-yVAD-CHO. (E) Caspase 1 activity was dose-dependently inhibited by AC-yVAD-CHO, and CSFV activated caspase 1 activity at an MOI of 1. * P

    Article Snippet: Primary antibodies used in the study included mouse monoclonal anti-CSFV E2 (JBT, 9011), mouse polyclonal anti-CSFV Npro (kindly provided by Dr. Xinglong Yu, Veterinary Department, Hunan Agricultural University, China), rabbit polyclonal anti-caspase-1 p10 (sc-514; Santa Cruz Biotechnology), mouse monoclonal anti-IL-1β (MP425; Thermo Fisher Scientific), rabbit polyclonal anti-IL-1β (ASC0912; Invitrogen), mouse monoclonal anti-GAPDH (AG019; Beyotime), mouse monoclonal anti-tubulin (AT819; Beyotime), rabbit monoclona anti-NLRP3 antibody (bs-23723R; Bioss), rabbit monoclona anti-ASC(bs-6741R; Bioss), rabbit polyclonal anti-Gasdermin D/DFNA5L(bs-14287R; Bioss), rabbit monoclonal anti-RIG-I antibody (D33H10; Cell Signaling Technology), and rabbit monoclonal anti- Caspase-1 antibody (D7F10; Cell Signaling Technology).The secondary antibodies included horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (BS12478; Bioworld), HRP-conjugated goat anti-rabbit IgG (BS13278; Bioworld), Dylight 488 goat anti-mouse IgG (E032210; EarthOx Life Sciences).

    Techniques: Activation Assay, Infection, Expressing, Western Blot, Activity Assay

    APOL1 risk alleles destabilize the adherens complex. ( A ) Protein blots of DPDs stably over-expressing Vector (V), G0, G1, and G2 were probed for APOL1, nephrin, CD2AP, dynamin, CTSL, and GAPDH (n = 5–7). Representative gels are displayed. ( B ) Protein blots of the above lysates were probed for dendrin and reprobed for GAPDH (n = 5). ( C – H ) Cumulative densitometric data on each variable (protein/GAPDH) are shown as dot plots. *

    Journal: Scientific Reports

    Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

    doi: 10.1038/s41598-019-39376-y

    Figure Lengend Snippet: APOL1 risk alleles destabilize the adherens complex. ( A ) Protein blots of DPDs stably over-expressing Vector (V), G0, G1, and G2 were probed for APOL1, nephrin, CD2AP, dynamin, CTSL, and GAPDH (n = 5–7). Representative gels are displayed. ( B ) Protein blots of the above lysates were probed for dendrin and reprobed for GAPDH (n = 5). ( C – H ) Cumulative densitometric data on each variable (protein/GAPDH) are shown as dot plots. *

    Article Snippet: Immunoprecipitation (IP) Lysates from control and experimental PDs were immunoprecipitated with either an anti-APOL1 (mouse monoclonal, Proteintech) - or an anti-CD2AP antibody (rabbit polyclonal; sc-25272, Santa Cruz).

    Techniques: Stable Transfection, Expressing, Plasmid Preparation

    Effect of Ectopic overexpression or silencing of APOL1 on the stability of adherens complexes. ( A ) PDs stably expressing vector (PDV) and ectopic APOL1G0 (PDG0) were differentiated for ten days and the transfected with either scrambled or APOL1 siRNA (n = 5). Protein blots were probed for APOL1, CD2AP, CTSL, and dynamin and reprobed for GAPDH. The Gels from two different lysates are displayed. ( B ) Protein blots from the above lysates were probed for nephrin and dendrin and reprobed for GAPDH. The gels from two different lysates are shown. ( C – H ) Densitometric data for each variable (protein/GAPDH) are shown as dot plots. ( C ) *P

    Journal: Scientific Reports

    Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

    doi: 10.1038/s41598-019-39376-y

    Figure Lengend Snippet: Effect of Ectopic overexpression or silencing of APOL1 on the stability of adherens complexes. ( A ) PDs stably expressing vector (PDV) and ectopic APOL1G0 (PDG0) were differentiated for ten days and the transfected with either scrambled or APOL1 siRNA (n = 5). Protein blots were probed for APOL1, CD2AP, CTSL, and dynamin and reprobed for GAPDH. The Gels from two different lysates are displayed. ( B ) Protein blots from the above lysates were probed for nephrin and dendrin and reprobed for GAPDH. The gels from two different lysates are shown. ( C – H ) Densitometric data for each variable (protein/GAPDH) are shown as dot plots. ( C ) *P

    Article Snippet: Immunoprecipitation (IP) Lysates from control and experimental PDs were immunoprecipitated with either an anti-APOL1 (mouse monoclonal, Proteintech) - or an anti-CD2AP antibody (rabbit polyclonal; sc-25272, Santa Cruz).

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

    An interaction between APOL1 and CD2AP at the plasma membrane. ( A ) Undifferentiated and differentiated PDG0 grown on coverslips were co-labeled for APOL1 and CD2AP. Representative fluoromicrographs are shown. Undifferentiated PDs displayed scattered labeling for APOL1 (red fluorescence) and CD2AP (green fluorescence). Differentiated PD showed co-labeling for APOL1 and CD2AP (yellow fluorescence, indicated by white arrows) at adherent junctions. Horizontal solid white bar represents the scale as 100 µm. ( B ) The CD2AP binds to the APOL1 in the carboxy-terminal region and residues from Membrane Addressing Domain (MAD) of APOL1 are also interacting with residues of CD2AP. Hotspot residues in the protein-protein interaction interface of APOL1G0 (Raspberry) and CD2AP (Warm pink). ( C ) The interaction interface of APOL1G0 and miR193a has a total interface area of 1339.1 Å 2 and the solvation free energy gain upon formation of interface Δ i G is −34.3 kcal/mol. The solvation free energy of folding for the corresponding structure ΔG is −263.1 kcal/mol. The hotspot residues (magenta) in the miRNA-Protein interaction interface of APOL1G0 and miR193a. The 5′ end of miR193a binds to the 3′ UTR region of APOL1G0 mRNA (36) and displayed symbolically on protein surface.

    Journal: Scientific Reports

    Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

    doi: 10.1038/s41598-019-39376-y

    Figure Lengend Snippet: An interaction between APOL1 and CD2AP at the plasma membrane. ( A ) Undifferentiated and differentiated PDG0 grown on coverslips were co-labeled for APOL1 and CD2AP. Representative fluoromicrographs are shown. Undifferentiated PDs displayed scattered labeling for APOL1 (red fluorescence) and CD2AP (green fluorescence). Differentiated PD showed co-labeling for APOL1 and CD2AP (yellow fluorescence, indicated by white arrows) at adherent junctions. Horizontal solid white bar represents the scale as 100 µm. ( B ) The CD2AP binds to the APOL1 in the carboxy-terminal region and residues from Membrane Addressing Domain (MAD) of APOL1 are also interacting with residues of CD2AP. Hotspot residues in the protein-protein interaction interface of APOL1G0 (Raspberry) and CD2AP (Warm pink). ( C ) The interaction interface of APOL1G0 and miR193a has a total interface area of 1339.1 Å 2 and the solvation free energy gain upon formation of interface Δ i G is −34.3 kcal/mol. The solvation free energy of folding for the corresponding structure ΔG is −263.1 kcal/mol. The hotspot residues (magenta) in the miRNA-Protein interaction interface of APOL1G0 and miR193a. The 5′ end of miR193a binds to the 3′ UTR region of APOL1G0 mRNA (36) and displayed symbolically on protein surface.

    Article Snippet: Immunoprecipitation (IP) Lysates from control and experimental PDs were immunoprecipitated with either an anti-APOL1 (mouse monoclonal, Proteintech) - or an anti-CD2AP antibody (rabbit polyclonal; sc-25272, Santa Cruz).

    Techniques: Labeling, Fluorescence

    APOL1 binds with one of the constituents of adherens complex. ( A ) Cellular lysates of DPDVs (DPDs expressing vector) and DPDG0s (DPDs expressing APOL1 G0) were probed for APOL1, CD2AP, dendrin, nephrin, and reprobed for actin (n = 3). Gels from three different lysates are displayed. ( B ) Cumulative densitometric data from protein blots displayed in 5A. **P

    Journal: Scientific Reports

    Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

    doi: 10.1038/s41598-019-39376-y

    Figure Lengend Snippet: APOL1 binds with one of the constituents of adherens complex. ( A ) Cellular lysates of DPDVs (DPDs expressing vector) and DPDG0s (DPDs expressing APOL1 G0) were probed for APOL1, CD2AP, dendrin, nephrin, and reprobed for actin (n = 3). Gels from three different lysates are displayed. ( B ) Cumulative densitometric data from protein blots displayed in 5A. **P

    Article Snippet: Immunoprecipitation (IP) Lysates from control and experimental PDs were immunoprecipitated with either an anti-APOL1 (mouse monoclonal, Proteintech) - or an anti-CD2AP antibody (rabbit polyclonal; sc-25272, Santa Cruz).

    Techniques: Expressing, Plasmid Preparation

    Evaluation of the effects of lack of APOL1 and Nephrin on the stability of adherens complex. ( A ) DPDS were transfected with either scrambled (SCR), APOL1- or nephrin-siRNAs (n = 10). Protein blots of control ( C ), DPD-transfected with SCR/SiRNA nephrin/siRNA APOL1 were probed for CD2AP, nephrin, APOL1, dendrin, and CTSL (n = 10). The same blots were reprobed for GAPDH. Gels from three different cellular lysates are displayed. ( B – F ) Cumulative densitometric data for each variable (protein/GAPDH) are shown as dot plots. ***p

    Journal: Scientific Reports

    Article Title: Disruption of APOL1-miR193a Axis Induces Disorganization of Podocyte Actin Cytoskeleton

    doi: 10.1038/s41598-019-39376-y

    Figure Lengend Snippet: Evaluation of the effects of lack of APOL1 and Nephrin on the stability of adherens complex. ( A ) DPDS were transfected with either scrambled (SCR), APOL1- or nephrin-siRNAs (n = 10). Protein blots of control ( C ), DPD-transfected with SCR/SiRNA nephrin/siRNA APOL1 were probed for CD2AP, nephrin, APOL1, dendrin, and CTSL (n = 10). The same blots were reprobed for GAPDH. Gels from three different cellular lysates are displayed. ( B – F ) Cumulative densitometric data for each variable (protein/GAPDH) are shown as dot plots. ***p

    Article Snippet: Immunoprecipitation (IP) Lysates from control and experimental PDs were immunoprecipitated with either an anti-APOL1 (mouse monoclonal, Proteintech) - or an anti-CD2AP antibody (rabbit polyclonal; sc-25272, Santa Cruz).

    Techniques: Transfection

    Western blotting results of Elk1 and Elk1-3R effects on both endogenous katanin-p60 and spastin proteins. (A) Analysis of SH-SY5Y transfection efficacy with His-tag including pCMV6_Elk1 and pCMV6_Elk1-3R vectors. (B) Western blotting results showing katanin-p60 level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (C) Quantification of katanin-p60 level was performed by normalizing band intensities of katanin-p60 to band intensities of ß-actin. Results indicated that both Elk1 and Elk1-3R decreased the katanin-p60 level significantly and the difference between them was not significant. SEM values are 0, 0.01, and 0.02, respectively (n = 4). (D) Western blotting results showing spastin level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (E) Quantification of spastin level was done as previously described. Results indicated that there was no significant spastin level difference between Elk1 and Elk1-3R transfected cells. However, they were significant compared to untransfected cells. SEM values are 0, 0.03, and 0.03, respectively (n = 4).

    Journal: PLoS ONE

    Article Title: Elk1 affects katanin and spastin proteins via differential transcriptional and post-transcriptional regulations

    doi: 10.1371/journal.pone.0212518

    Figure Lengend Snippet: Western blotting results of Elk1 and Elk1-3R effects on both endogenous katanin-p60 and spastin proteins. (A) Analysis of SH-SY5Y transfection efficacy with His-tag including pCMV6_Elk1 and pCMV6_Elk1-3R vectors. (B) Western blotting results showing katanin-p60 level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (C) Quantification of katanin-p60 level was performed by normalizing band intensities of katanin-p60 to band intensities of ß-actin. Results indicated that both Elk1 and Elk1-3R decreased the katanin-p60 level significantly and the difference between them was not significant. SEM values are 0, 0.01, and 0.02, respectively (n = 4). (D) Western blotting results showing spastin level in untransfected (control), pCMV6_Elk1 transfected, and pCMV6_Elk1-3R transfected SH-SY5Y cells. Transfection experiment was performed two times on separate days and Western blotting experiment was performed two times with each sample. (E) Quantification of spastin level was done as previously described. Results indicated that there was no significant spastin level difference between Elk1 and Elk1-3R transfected cells. However, they were significant compared to untransfected cells. SEM values are 0, 0.03, and 0.03, respectively (n = 4).

    Article Snippet: Membrane was blocked in 5% skim milk powder/TBST (Tris buffered saline (TBS) containing 0.1% Tween 20) for 1 h at room temperature, and then incubated with the following primary antibodies at indicated dilutions overnight at 4°C; rabbit monoclonal His-tag antibody (1:1000, Cell Signaling Technology), mouse monoclonal spastin antibody (1:1000, Sigma), rabbit polyclonal katanin-p60 antibody (1:1000, ATLAS), mouse monoclonal p27 antibody (1:500, Santa Cruz), mouse monoclonal HuR antibody (1:500, Santa Cruz), mouse monoclonal β-Actin antibody (1:1000, Cell Signaling Technology), rabbit monoclonal GAPDH antibody (1:1000, Cell Signaling Technology), rabbit monoclonal β-tubulin antibody (1:1000, Cell Signaling Technology) in 5% skim milk powder/TBST.

    Techniques: Western Blot, Transfection

    ICC results of Elk1 and Elk1-3R effects on endogenous katanin-p60 level. Asterisk symbol represents untransfected cells, whereas the cells indicated with arrows are either Elk1 or Elk1-3R overexpressing cells. The images were taken using 63X objective at zoom 2.6. (A) The level of endogenous katanin-p60 protein is reduced in Elk1 overexpressed SH-SY5Y cells compared to untransfected cells. (B) Relative fluorescence density graph shows that the expression of katanin-p60 was 5.5 fold decreased in Elk1 overexpressed cells compared to control cells. SEM values are 0 and 0.02, respectively. (C) The decrease in endogenous katanin-p60 level is observed in Elk1-3R overexpressed SH-SY5Y cells compared to untransfected cells. (D) Relative fluorescence density graph shows that the expression of katanin-p60 was 4.76 fold decreased in Elk1-3R overexpressed cells compared to control cells. SEM values are 0 and 0.03, respectively.

    Journal: PLoS ONE

    Article Title: Elk1 affects katanin and spastin proteins via differential transcriptional and post-transcriptional regulations

    doi: 10.1371/journal.pone.0212518

    Figure Lengend Snippet: ICC results of Elk1 and Elk1-3R effects on endogenous katanin-p60 level. Asterisk symbol represents untransfected cells, whereas the cells indicated with arrows are either Elk1 or Elk1-3R overexpressing cells. The images were taken using 63X objective at zoom 2.6. (A) The level of endogenous katanin-p60 protein is reduced in Elk1 overexpressed SH-SY5Y cells compared to untransfected cells. (B) Relative fluorescence density graph shows that the expression of katanin-p60 was 5.5 fold decreased in Elk1 overexpressed cells compared to control cells. SEM values are 0 and 0.02, respectively. (C) The decrease in endogenous katanin-p60 level is observed in Elk1-3R overexpressed SH-SY5Y cells compared to untransfected cells. (D) Relative fluorescence density graph shows that the expression of katanin-p60 was 4.76 fold decreased in Elk1-3R overexpressed cells compared to control cells. SEM values are 0 and 0.03, respectively.

    Article Snippet: Membrane was blocked in 5% skim milk powder/TBST (Tris buffered saline (TBS) containing 0.1% Tween 20) for 1 h at room temperature, and then incubated with the following primary antibodies at indicated dilutions overnight at 4°C; rabbit monoclonal His-tag antibody (1:1000, Cell Signaling Technology), mouse monoclonal spastin antibody (1:1000, Sigma), rabbit polyclonal katanin-p60 antibody (1:1000, ATLAS), mouse monoclonal p27 antibody (1:500, Santa Cruz), mouse monoclonal HuR antibody (1:500, Santa Cruz), mouse monoclonal β-Actin antibody (1:1000, Cell Signaling Technology), rabbit monoclonal GAPDH antibody (1:1000, Cell Signaling Technology), rabbit monoclonal β-tubulin antibody (1:1000, Cell Signaling Technology) in 5% skim milk powder/TBST.

    Techniques: Immunocytochemistry, Fluorescence, Expressing