ago2 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ago2 antibodies
    CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against <t>AGO2</t> on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.
    Ago2 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/EZH2 axis"

    Article Title: Circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/EZH2 axis

    Journal: Theranostics

    doi: 10.7150/thno.42417

    CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against AGO2 on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.
    Figure Legend Snippet: CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against AGO2 on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.

    Techniques Used: Quantitative RT-PCR, RNA In Situ Hybridization, Staining, Luciferase, Transfection, Plasmid Preparation, Negative Control, Expressing

    ago2 antibodies  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ago2 antibodies
    CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against <t>AGO2</t> on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.
    Ago2 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/EZH2 axis"

    Article Title: Circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/EZH2 axis

    Journal: Theranostics

    doi: 10.7150/thno.42417

    CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against AGO2 on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.
    Figure Legend Snippet: CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against AGO2 on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.

    Techniques Used: Quantitative RT-PCR, RNA In Situ Hybridization, Staining, Luciferase, Transfection, Plasmid Preparation, Negative Control, Expressing

    ago2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ago2 antibody
    LINC0086 interacts with miR-214 and regulates its expression. (A) qPCR was performed to detect the expression of miR-214 in C666-1 and HK-1 cells after LINC0086 transfection. (B) The wild type (WT) and mutant type (MT) of LINC0086 3′-UTR, and the binding sites were shown. (C) The relative luciferase activities were inhibited in the C666-1 and HK-1 cells cotransfected with wild-type LINC0086 3′-UTR and miR-214, and not with the mutant type. Firefly luciferase activity was normalized to Renilla luciferase. (D) Association of LINC0086 and miR-214 with <t>Ago2</t> in C666-1 and HK-1 cells. Cellular lysates from C666-1 and HK-1 cells were used for RNA immunoprecipitation (RIP) with antibody against Ago2. LINC0086 and miR-214 expression levels were detected using qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group.
    Ago2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Long Noncoding RNA LINC0086 Functions as a Tumor Suppressor in Nasopharyngeal Carcinoma by Targeting miR-214"

    Article Title: Long Noncoding RNA LINC0086 Functions as a Tumor Suppressor in Nasopharyngeal Carcinoma by Targeting miR-214

    Journal: Oncology Research

    doi: 10.3727/096504017X14865126670075

    LINC0086 interacts with miR-214 and regulates its expression. (A) qPCR was performed to detect the expression of miR-214 in C666-1 and HK-1 cells after LINC0086 transfection. (B) The wild type (WT) and mutant type (MT) of LINC0086 3′-UTR, and the binding sites were shown. (C) The relative luciferase activities were inhibited in the C666-1 and HK-1 cells cotransfected with wild-type LINC0086 3′-UTR and miR-214, and not with the mutant type. Firefly luciferase activity was normalized to Renilla luciferase. (D) Association of LINC0086 and miR-214 with Ago2 in C666-1 and HK-1 cells. Cellular lysates from C666-1 and HK-1 cells were used for RNA immunoprecipitation (RIP) with antibody against Ago2. LINC0086 and miR-214 expression levels were detected using qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group.
    Figure Legend Snippet: LINC0086 interacts with miR-214 and regulates its expression. (A) qPCR was performed to detect the expression of miR-214 in C666-1 and HK-1 cells after LINC0086 transfection. (B) The wild type (WT) and mutant type (MT) of LINC0086 3′-UTR, and the binding sites were shown. (C) The relative luciferase activities were inhibited in the C666-1 and HK-1 cells cotransfected with wild-type LINC0086 3′-UTR and miR-214, and not with the mutant type. Firefly luciferase activity was normalized to Renilla luciferase. (D) Association of LINC0086 and miR-214 with Ago2 in C666-1 and HK-1 cells. Cellular lysates from C666-1 and HK-1 cells were used for RNA immunoprecipitation (RIP) with antibody against Ago2. LINC0086 and miR-214 expression levels were detected using qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group.

    Techniques Used: Expressing, Transfection, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Immunoprecipitation, Quantitative RT-PCR

    ani ago2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ani ago2 antibody
    CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the <t>Ago2</t> or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.
    Ani Ago2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ani ago2 antibody - by Bioz Stars, 2023-02
    95/100 stars

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    1) Product Images from "Arterial cyclic stretch regulates Lamtor1 and promotes neointimal hyperplasia via circSlc8a1/miR-20a-5p axis in vein grafts"

    Article Title: Arterial cyclic stretch regulates Lamtor1 and promotes neointimal hyperplasia via circSlc8a1/miR-20a-5p axis in vein grafts

    Journal: Theranostics

    doi: 10.7150/thno.69551

    CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the Ago2 or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.
    Figure Legend Snippet: CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the Ago2 or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.

    Techniques Used: Expressing, Western Blot, Negative Control, Immunoprecipitation, Quantitative RT-PCR, Luciferase, Reporter Assay, Mutagenesis, Transfection, Enzyme-linked Immunosorbent Assay

    antibody against ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antibody against ago2
    Antibody Against Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ago2  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ago2
    ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with <t>AGO2</t> antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).
    Ago2, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "CREB3 Transactivates lncRNA ZFAS1 to Promote Papillary Thyroid Carcinoma Metastasis by Modulating miR-373-3 p /MMP3 Regulatory Axis"

    Article Title: CREB3 Transactivates lncRNA ZFAS1 to Promote Papillary Thyroid Carcinoma Metastasis by Modulating miR-373-3 p /MMP3 Regulatory Axis

    Journal: International Journal of Endocrinology

    doi: 10.1155/2021/9981683

    ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with AGO2 antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).
    Figure Legend Snippet: ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with AGO2 antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).

    Techniques Used: Luciferase, Expressing, Immunoprecipitation

    ago2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ago2 antibody
    Ago2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ago2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ago2 antibody
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    anti ago2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti ago2 antibody
    Anti Ago2 Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ago2 antibody  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc ago2 antibody
    CircDLG1 functions as a sponge for miR-141-3p to regulate CXCL12 expression. a The results of RNA immunoprecipitation for <t>AGO2</t> in SGC7901 cells stably expressing Flag-AGO2 and Flag-GFP. ** P < 0.01, Student’s t-test; the experiment was repeated three times. b The luciferase activity of the circDLG1 reporter after transfection of circDLG1 shRNA in SGC7901 cells. * P < 0.05, Student’s t-test; the experiment was repeated three times. c A luciferase screen with a miRNA mimic library was constructed in SGC7901 cells. d The luciferase activity of the circDLG1 reporter after transfection with four miRNAs (miR-141-3p, miR-138-5p, miR-651-3p, and miR-155-3p) in SGC7901 cells. * P < 0.05, one-way ANOVA test for the comparison of all groups, followed by Student’s t-test for the comparison of miR-138-5p, miR-141-3p, miR-651-3p, and miR-155-3p with the control group. Multigroup comparisons were adjusted using the Bonferroni method. The experiment was repeated three times. e RIP analysis with a biotin-labeled circDLG1 probe in SGC7901 cells. * P < 0.05, Student’s t-test; the experiment was repeated three times. f RIP analysis with a biotin-labeled circDLG1 probe in SGC7901 cells treated with sh-circDLG1. * P < 0.05, Student’s t-test; the experiment was repeated three times. g The association between the expression of circDLG1 and miR-141-3p in gastric cancer tissues. n = 82, P < 0.001, Pearson correlation analysis. h RNA FISH assay of circDLG1 and miR-141-3p in gastric cancer cells. Scale bar, 50 μm. i The mRNA level of CXCL12 in gastric cancer cells after the expression of miR-141-3p. * P < 0.05, Student’s t-test; the experiment was repeated three times. j SGC7901 cells and SGC7901 cells with circDLG1 knockdown (SGC7901-sh-circDLG1#1/SGC7901-sh-NC) were treated with miR-NC, miR-141-3p, or miR-141-3p inhibitor, the protein was extracted, and western blot analysis of CXCL12 protein levels was performed. k SGC7901 cells and SGC7901 cells with circDLG1 knockdown (SGC7901-sh-circDLG1#1/SGC7901-sh-NC) were cotransfected with miR-NC, miR-141-3p, or miR-141-3p inhibitor and pGL-luc-CXCL12 3′-UTR (wild-type) or pGL-luc-CXCL12 3′-UTR (mutant-type). Luciferase activity was detected using the dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions. * P < 0.05, one-way ANOVA test for the comparison of all groups, followed by Student’s t-test for the comparison of the other groups with the sh-NC + miR-NC group. Multigroup comparisons were adjusted using the Bonferroni method. The experiment was repeated three times. l The association between miR-141-3p expression and CXCL12 expression in gastric cancer tissues. n = 82, P < 0.001, Pearson correlation analysis
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    1) Product Images from "The circular RNA circDLG1 promotes gastric cancer progression and anti-PD-1 resistance through the regulation of CXCL12 by sponging miR-141-3p"

    Article Title: The circular RNA circDLG1 promotes gastric cancer progression and anti-PD-1 resistance through the regulation of CXCL12 by sponging miR-141-3p

    Journal: Molecular Cancer

    doi: 10.1186/s12943-021-01475-8

    CircDLG1 functions as a sponge for miR-141-3p to regulate CXCL12 expression. a The results of RNA immunoprecipitation for AGO2 in SGC7901 cells stably expressing Flag-AGO2 and Flag-GFP. ** P < 0.01, Student’s t-test; the experiment was repeated three times. b The luciferase activity of the circDLG1 reporter after transfection of circDLG1 shRNA in SGC7901 cells. * P < 0.05, Student’s t-test; the experiment was repeated three times. c A luciferase screen with a miRNA mimic library was constructed in SGC7901 cells. d The luciferase activity of the circDLG1 reporter after transfection with four miRNAs (miR-141-3p, miR-138-5p, miR-651-3p, and miR-155-3p) in SGC7901 cells. * P < 0.05, one-way ANOVA test for the comparison of all groups, followed by Student’s t-test for the comparison of miR-138-5p, miR-141-3p, miR-651-3p, and miR-155-3p with the control group. Multigroup comparisons were adjusted using the Bonferroni method. The experiment was repeated three times. e RIP analysis with a biotin-labeled circDLG1 probe in SGC7901 cells. * P < 0.05, Student’s t-test; the experiment was repeated three times. f RIP analysis with a biotin-labeled circDLG1 probe in SGC7901 cells treated with sh-circDLG1. * P < 0.05, Student’s t-test; the experiment was repeated three times. g The association between the expression of circDLG1 and miR-141-3p in gastric cancer tissues. n = 82, P < 0.001, Pearson correlation analysis. h RNA FISH assay of circDLG1 and miR-141-3p in gastric cancer cells. Scale bar, 50 μm. i The mRNA level of CXCL12 in gastric cancer cells after the expression of miR-141-3p. * P < 0.05, Student’s t-test; the experiment was repeated three times. j SGC7901 cells and SGC7901 cells with circDLG1 knockdown (SGC7901-sh-circDLG1#1/SGC7901-sh-NC) were treated with miR-NC, miR-141-3p, or miR-141-3p inhibitor, the protein was extracted, and western blot analysis of CXCL12 protein levels was performed. k SGC7901 cells and SGC7901 cells with circDLG1 knockdown (SGC7901-sh-circDLG1#1/SGC7901-sh-NC) were cotransfected with miR-NC, miR-141-3p, or miR-141-3p inhibitor and pGL-luc-CXCL12 3′-UTR (wild-type) or pGL-luc-CXCL12 3′-UTR (mutant-type). Luciferase activity was detected using the dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions. * P < 0.05, one-way ANOVA test for the comparison of all groups, followed by Student’s t-test for the comparison of the other groups with the sh-NC + miR-NC group. Multigroup comparisons were adjusted using the Bonferroni method. The experiment was repeated three times. l The association between miR-141-3p expression and CXCL12 expression in gastric cancer tissues. n = 82, P < 0.001, Pearson correlation analysis
    Figure Legend Snippet: CircDLG1 functions as a sponge for miR-141-3p to regulate CXCL12 expression. a The results of RNA immunoprecipitation for AGO2 in SGC7901 cells stably expressing Flag-AGO2 and Flag-GFP. ** P < 0.01, Student’s t-test; the experiment was repeated three times. b The luciferase activity of the circDLG1 reporter after transfection of circDLG1 shRNA in SGC7901 cells. * P < 0.05, Student’s t-test; the experiment was repeated three times. c A luciferase screen with a miRNA mimic library was constructed in SGC7901 cells. d The luciferase activity of the circDLG1 reporter after transfection with four miRNAs (miR-141-3p, miR-138-5p, miR-651-3p, and miR-155-3p) in SGC7901 cells. * P < 0.05, one-way ANOVA test for the comparison of all groups, followed by Student’s t-test for the comparison of miR-138-5p, miR-141-3p, miR-651-3p, and miR-155-3p with the control group. Multigroup comparisons were adjusted using the Bonferroni method. The experiment was repeated three times. e RIP analysis with a biotin-labeled circDLG1 probe in SGC7901 cells. * P < 0.05, Student’s t-test; the experiment was repeated three times. f RIP analysis with a biotin-labeled circDLG1 probe in SGC7901 cells treated with sh-circDLG1. * P < 0.05, Student’s t-test; the experiment was repeated three times. g The association between the expression of circDLG1 and miR-141-3p in gastric cancer tissues. n = 82, P < 0.001, Pearson correlation analysis. h RNA FISH assay of circDLG1 and miR-141-3p in gastric cancer cells. Scale bar, 50 μm. i The mRNA level of CXCL12 in gastric cancer cells after the expression of miR-141-3p. * P < 0.05, Student’s t-test; the experiment was repeated three times. j SGC7901 cells and SGC7901 cells with circDLG1 knockdown (SGC7901-sh-circDLG1#1/SGC7901-sh-NC) were treated with miR-NC, miR-141-3p, or miR-141-3p inhibitor, the protein was extracted, and western blot analysis of CXCL12 protein levels was performed. k SGC7901 cells and SGC7901 cells with circDLG1 knockdown (SGC7901-sh-circDLG1#1/SGC7901-sh-NC) were cotransfected with miR-NC, miR-141-3p, or miR-141-3p inhibitor and pGL-luc-CXCL12 3′-UTR (wild-type) or pGL-luc-CXCL12 3′-UTR (mutant-type). Luciferase activity was detected using the dual-luciferase reporter assay system (Promega, USA) according to the manufacturer’s instructions. * P < 0.05, one-way ANOVA test for the comparison of all groups, followed by Student’s t-test for the comparison of the other groups with the sh-NC + miR-NC group. Multigroup comparisons were adjusted using the Bonferroni method. The experiment was repeated three times. l The association between miR-141-3p expression and CXCL12 expression in gastric cancer tissues. n = 82, P < 0.001, Pearson correlation analysis

    Techniques Used: Expressing, Immunoprecipitation, Stable Transfection, Luciferase, Activity Assay, Transfection, shRNA, Construct, Labeling, Western Blot, Mutagenesis, Reporter Assay

    antiargonaute 1  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc antiargonaute 1
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    Cell Signaling Technology Inc ago2 antibodies
    CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against <t>AGO2</t> on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.
    Ago2 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc ago2 antibody
    LINC0086 interacts with miR-214 and regulates its expression. (A) qPCR was performed to detect the expression of miR-214 in C666-1 and HK-1 cells after LINC0086 transfection. (B) The wild type (WT) and mutant type (MT) of LINC0086 3′-UTR, and the binding sites were shown. (C) The relative luciferase activities were inhibited in the C666-1 and HK-1 cells cotransfected with wild-type LINC0086 3′-UTR and miR-214, and not with the mutant type. Firefly luciferase activity was normalized to Renilla luciferase. (D) Association of LINC0086 and miR-214 with <t>Ago2</t> in C666-1 and HK-1 cells. Cellular lysates from C666-1 and HK-1 cells were used for RNA immunoprecipitation (RIP) with antibody against Ago2. LINC0086 and miR-214 expression levels were detected using qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group.
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    Cell Signaling Technology Inc ani ago2 antibody
    CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the <t>Ago2</t> or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.
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    Cell Signaling Technology Inc antibody against ago2
    CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the <t>Ago2</t> or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.
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    ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with <t>AGO2</t> antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).
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    ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with <t>AGO2</t> antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).
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    Cell Signaling Technology Inc antiargonaute 1
    ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with <t>AGO2</t> antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).
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    Image Search Results


    CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against AGO2 on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.

    Journal: Theranostics

    Article Title: Circular RNA SAMD4A controls adipogenesis in obesity through the miR-138-5p/EZH2 axis

    doi: 10.7150/thno.42417

    Figure Lengend Snippet: CircSAMD4A acts as a miRNA sponge for miR-138-5p. (A) CircSAMD4A-miRNA-mRNA network and pathway analysis. (B) RIP experiments were performed using an antibody against AGO2 on extracts from preadipocytes. (C) CircRIP was performed using a circSAMD4A-specific probe and control probe in preadipocytes from obese patients. The enrichment of circSAMD4A and microRNAs was detected by qRT-PCR and normalized to the control probe. (D) Co-localization between circSAMD4A and miR-138-5p was observed by RNA in situ hybridization in preadipocytes. Nuclei were stained with DAPI. Scale bar = 20µm. (E) Schematic showing the predicted miR-138-5p sites in circSAMD4A. (F) Luciferase assays in preadipocytes co-transfected with a scrambled control, miR-138-5p mimic, and a luciferase reporter plasmid containing either wild-type circSAMD4A (circSAMD4A-WT). (G) qRT-PCR showing the level of circSAMD4A in the streptavidin-captured fractions from the preadipocytes lysates after transfection with biotinylated miR-138-5p or control RNA. CircANRIL (a circular RNA reported not to bind to AGO2) was used as a negative control. (H) Pearson correlation between circSAMD4A expression and miR-138-5p expression in adipose tissues of 60 obese patients using qRT-PCR. Data are presented as means ± SD; significant difference was identified with Student's t test. *P < 0.05; **P < 0.01.

    Article Snippet: After blocking for 1 h, the membranes were incubated with the primary anti-EZH2, C/EBP-α, PPAR-γ or AGO2 antibodies (1:000; Cell Signaling Technology, Danvers, MA, USA) at 4ºC overnight, followed by a 2 h incubation with HRP-conjugated secondary antibody at room temperature.

    Techniques: Quantitative RT-PCR, RNA In Situ Hybridization, Staining, Luciferase, Transfection, Plasmid Preparation, Negative Control, Expressing

    LINC0086 interacts with miR-214 and regulates its expression. (A) qPCR was performed to detect the expression of miR-214 in C666-1 and HK-1 cells after LINC0086 transfection. (B) The wild type (WT) and mutant type (MT) of LINC0086 3′-UTR, and the binding sites were shown. (C) The relative luciferase activities were inhibited in the C666-1 and HK-1 cells cotransfected with wild-type LINC0086 3′-UTR and miR-214, and not with the mutant type. Firefly luciferase activity was normalized to Renilla luciferase. (D) Association of LINC0086 and miR-214 with Ago2 in C666-1 and HK-1 cells. Cellular lysates from C666-1 and HK-1 cells were used for RNA immunoprecipitation (RIP) with antibody against Ago2. LINC0086 and miR-214 expression levels were detected using qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group.

    Journal: Oncology Research

    Article Title: Long Noncoding RNA LINC0086 Functions as a Tumor Suppressor in Nasopharyngeal Carcinoma by Targeting miR-214

    doi: 10.3727/096504017X14865126670075

    Figure Lengend Snippet: LINC0086 interacts with miR-214 and regulates its expression. (A) qPCR was performed to detect the expression of miR-214 in C666-1 and HK-1 cells after LINC0086 transfection. (B) The wild type (WT) and mutant type (MT) of LINC0086 3′-UTR, and the binding sites were shown. (C) The relative luciferase activities were inhibited in the C666-1 and HK-1 cells cotransfected with wild-type LINC0086 3′-UTR and miR-214, and not with the mutant type. Firefly luciferase activity was normalized to Renilla luciferase. (D) Association of LINC0086 and miR-214 with Ago2 in C666-1 and HK-1 cells. Cellular lysates from C666-1 and HK-1 cells were used for RNA immunoprecipitation (RIP) with antibody against Ago2. LINC0086 and miR-214 expression levels were detected using qRT-PCR. * p < 0.05, ** p < 0.01, *** p < 0.001 versus NC group.

    Article Snippet: RIP experiments were performed using the Magna RIP RNA-Binding Protein IP Kit (Millipore, Bedford, MA, USA) and the Ago2 antibody (Cell Signaling, Danvers, MA, USA) according to the manufacturer’s instructions.

    Techniques: Expressing, Transfection, Mutagenesis, Binding Assay, Luciferase, Activity Assay, Immunoprecipitation, Quantitative RT-PCR

    CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the Ago2 or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.

    Journal: Theranostics

    Article Title: Arterial cyclic stretch regulates Lamtor1 and promotes neointimal hyperplasia via circSlc8a1/miR-20a-5p axis in vein grafts

    doi: 10.7150/thno.69551

    Figure Lengend Snippet: CircSlc8a1 positively regulated Lamtor1 expression and venous SMC dedifferentiation and proliferation via sponging for miR-20a-5p. A. Western blot indicated that in venous SMCs, inhibitor of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p increased Lamtor1 expression compared with the negative control (NC) ( n = 4). B. In venous SMCs, mimics of miR-17-5p, miR-20a-5p, miR-29a-3p or miR-29c-3p decreased Lamtor1 expression compared with NC ( n = 4). C. Among the 4 miRNA candidates, only miR-20a-5p was significantly pulled down with circSlc8a1 biotin-coupled probe in lysates of venous SMCs. D. RIP assay was conducted using the Ago2 or IgG antibody for immunoprecipitation. The expressions of circSlc8a1 and miR-20a-5p were detected by RT-qPCR in venous SMCs ( n = 3). E. Luciferase reporter assay with wild type (WT) or mutant (Mut) circSlc8a1 co-transfected with miR-20a-5p mimics or nonsilencing control (NC). Co-tranfection of WT circSlc8a1 and miR-20a-5p significantly reduced luciferase levels ( n = 3). F. The repression of circSlc8a1 inhibited the protein level of Lamtor1, which could be abolished by miR-20a-5p silencing ( n = 5). G. Proliferation of venous SMCs was detected by BrdU-ELISA after transfected with circSlc8a1 specific siRNA (sicircSlc8a1) and simultaneous transfection of miR-20a-5p inhibitor (IN), respectively ( n = 5). H. The expressions of venous SMC differentiation makers were determined by western blot after treated with Rapamycin (50 nM) ( n = 5). I. The expressions of Lamtor1 and differentiation makers were determined by western blot after transfection of Lamtor1 specific siRNA ( n = 5). J. BrdU-ELISA revealed that specific Lamtor1 siRNAs decreased venous SMC proliferation compared with nonsilencing control (NC) ( n = 5). K. The expressions of α-SMA, calponin and SM22 were detected by western blot after treated with leucine and the combination with Lamtor1-specific siRNA (siLam) or circSlc8a1-specific siRNA (sicircSlc) ( n = 5). Data were represented as mean ± SD . ✳ P < 0.05, ØØ P < 0.01, ØØØ P < 0.001.

    Article Snippet: Then the cell lysis was incubated with magnetic beads conjugated with ani-Ago2 antibody (CST) or negative control IgG, at 4 °C overnight.

    Techniques: Expressing, Western Blot, Negative Control, Immunoprecipitation, Quantitative RT-PCR, Luciferase, Reporter Assay, Mutagenesis, Transfection, Enzyme-linked Immunosorbent Assay

    ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with AGO2 antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).

    Journal: International Journal of Endocrinology

    Article Title: CREB3 Transactivates lncRNA ZFAS1 to Promote Papillary Thyroid Carcinoma Metastasis by Modulating miR-373-3 p /MMP3 Regulatory Axis

    doi: 10.1155/2021/9981683

    Figure Lengend Snippet: ZFAS1 modulated miR-373-3 p /MMP3 axis. (a) MMP3 was predicted as a direct target of miR-373-3 p . (b) Target between MMP3 and miR-373-3 p was confirmed by luciferase assay ( ∗∗ p < 0.01). (c) The effect of miR-373-3 p on MMP3 expression in TPC-1 cell was tested by q-PCR ( ∗∗ p < 0.01). (d) MMP3 protein level was decreased by miR-373-3 p mimics ( ∗∗ p < 0.01). (e) miR-373-3 p and MMP3 expression were tested by q-PCR after ZFAS1 knocking down in TPC-1 cells ( ∗∗ p < 0.01, ∗∗∗ p < 0.001). (f) MMP3 expression profile in primary tumor and normal tissues obtained from TCGA-THCA datasets ( ∗∗∗ p < 0.001). (g) The effect of MMP3 knocking down on ZFAS1 expression was tested by q-PCR. (h) ZFAS1 and MMP3 enrichment with AGO2 antibody analyzed by RNA immunoprecipitation followed q-PCR test after ectopically expressed miR-373-3 p ( ∗∗ p < 0.01).

    Article Snippet: AGO2 (anti-AGO2, 1 : 50, Cell Signaling, USA) antibody or IgG was used and immunoprecipitated by 25 μ l protein A/G ligated beads.

    Techniques: Luciferase, Expressing, Immunoprecipitation