Structured Review

Cell Signaling Technology Inc mouse anti sp1
EMSA of (A) I <t>Sp1</t> (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.
Mouse Anti Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/mouse anti sp1/product/Cell Signaling Technology Inc
Average 88 stars, based on 1 article reviews
Price from $9.99 to $1999.99
mouse anti sp1 - by Bioz Stars, 2020-08
88/100 stars

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1) Product Images from "Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function"

Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

Journal: PLoS ONE

doi: 10.1371/journal.pone.0005049

EMSA of (A) I Sp1 (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.
Figure Legend Snippet: EMSA of (A) I Sp1 (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.

Techniques Used: Labeling, Incubation, Binding Assay, Chromatin Immunoprecipitation, Isolation, Polymerase Chain Reaction

Characterization of SPAK promoter. A. Schematic representation of human SPAK promoter constructs. the full-length SPAK promoter (nt-1472 to +4); construct I (nt −1050 to +4); construct II (nt −398 to +4); construct III (nt −331 to +4); construct IV (nt −149 to +4) and construct V (nt −72 to +4). Numbers are given in relation to the translational start codon (+1) and indicate 5′-ends of the deletion constructs. The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 (Red) and NF-κB (Yellow) sites are indicated by arrows. B. Promoter activities of the 5′ deleted constructs in un-treated or hyperosmolarity-stimulated Caco2-BBE cells normalized to Renilla Luc activities driven by the phRL-CMV control vector. Activities are expressed as fold inductions over cells transfected with the empty pGL3-basic vector. Each value represents the mean±SD of at least 3 independent sets of transfection experiments performed in triplicate, *p
Figure Legend Snippet: Characterization of SPAK promoter. A. Schematic representation of human SPAK promoter constructs. the full-length SPAK promoter (nt-1472 to +4); construct I (nt −1050 to +4); construct II (nt −398 to +4); construct III (nt −331 to +4); construct IV (nt −149 to +4) and construct V (nt −72 to +4). Numbers are given in relation to the translational start codon (+1) and indicate 5′-ends of the deletion constructs. The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 (Red) and NF-κB (Yellow) sites are indicated by arrows. B. Promoter activities of the 5′ deleted constructs in un-treated or hyperosmolarity-stimulated Caco2-BBE cells normalized to Renilla Luc activities driven by the phRL-CMV control vector. Activities are expressed as fold inductions over cells transfected with the empty pGL3-basic vector. Each value represents the mean±SD of at least 3 independent sets of transfection experiments performed in triplicate, *p

Techniques Used: Construct, Plasmid Preparation, Transfection

Western blots of transcription factors Sp1 and NF-κB (p65). A. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vivo . Histone3 acts as a control. B. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vitro . Histone3 acts as a control. C. Reduction of NF-κB but not Sp1 expression reduced SPAK protein expression in unstimulated and in hyperosmolarity-stimulated Caco2-BBE cells. Cells were harvested and subjected to western blot analysis using Sp1, NF-κB (p65), and SPAK antibodies as described in materials and methods . GAPDH acts as a loading control.
Figure Legend Snippet: Western blots of transcription factors Sp1 and NF-κB (p65). A. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vivo . Histone3 acts as a control. B. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vitro . Histone3 acts as a control. C. Reduction of NF-κB but not Sp1 expression reduced SPAK protein expression in unstimulated and in hyperosmolarity-stimulated Caco2-BBE cells. Cells were harvested and subjected to western blot analysis using Sp1, NF-κB (p65), and SPAK antibodies as described in materials and methods . GAPDH acts as a loading control.

Techniques Used: Western Blot, In Vivo, In Vitro, Expressing

Related Articles

Immunoprecipitation:

Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function
Article Snippet: .. Samples were then immunoprecipitated with 2 μg of mouse anti-Sp1 (Upstate Cell Signaling Solutions) or 3 μg of rabbit anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C. ..

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    Cell Signaling Technology Inc sp1 antibodies
    Overexpression of <t>Sp1</t> impairs miR-29c-induced inhibition of EMT The mRNA and protein expression of TGF-β1-induced EMT-associated markers including TTF, E-cadherin, vimentin and α-SMA were analyzed in ( A – B ) 95C and ( C – D ) A549 cells with ectopically expressing miR-29c mimics or pcDNA-Sp1. * p
    Sp1 Antibodies, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc mouse anti sp1
    EMSA of (A) I <t>Sp1</t> (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.
    Mouse Anti Sp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mouse anti sp1/product/Cell Signaling Technology Inc
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mouse anti sp1 - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

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    Overexpression of Sp1 impairs miR-29c-induced inhibition of EMT The mRNA and protein expression of TGF-β1-induced EMT-associated markers including TTF, E-cadherin, vimentin and α-SMA were analyzed in ( A – B ) 95C and ( C – D ) A549 cells with ectopically expressing miR-29c mimics or pcDNA-Sp1. * p

    Journal: Oncotarget

    Article Title: A regulatory loop involving miR-29c and Sp1 elevates the TGF-β1 mediated epithelial-to-mesenchymal transition in lung cancer

    doi: 10.18632/oncotarget.13137

    Figure Lengend Snippet: Overexpression of Sp1 impairs miR-29c-induced inhibition of EMT The mRNA and protein expression of TGF-β1-induced EMT-associated markers including TTF, E-cadherin, vimentin and α-SMA were analyzed in ( A – B ) 95C and ( C – D ) A549 cells with ectopically expressing miR-29c mimics or pcDNA-Sp1. * p

    Article Snippet: E-Cadherin, TIF-1, vimentin, a-SMA and sp1 antibodies were obtained from Cell Signaling Tech (Denver, MA).

    Techniques: Over Expression, Inhibition, Expressing

    Summary diagram describes the miR-29c/Sp1 network that regulates TGF-β1 expression and TGF-β1-induced EMT

    Journal: Oncotarget

    Article Title: A regulatory loop involving miR-29c and Sp1 elevates the TGF-β1 mediated epithelial-to-mesenchymal transition in lung cancer

    doi: 10.18632/oncotarget.13137

    Figure Lengend Snippet: Summary diagram describes the miR-29c/Sp1 network that regulates TGF-β1 expression and TGF-β1-induced EMT

    Article Snippet: E-Cadherin, TIF-1, vimentin, a-SMA and sp1 antibodies were obtained from Cell Signaling Tech (Denver, MA).

    Techniques: Expressing

    Inhibition of miR-29c significantly elevates the migration and invasion ( A ) The expression of mir-29c and Sp1 in TGF-β1-treated cells. Transwell assay was performed and ( B – C ) the migration and invasion of 95C and A549 cells transfected with miR-29c inhibitors were determined. ( D ) The evaluation of transfection efficiency of miR-29c inhibitors. * p

    Journal: Oncotarget

    Article Title: A regulatory loop involving miR-29c and Sp1 elevates the TGF-β1 mediated epithelial-to-mesenchymal transition in lung cancer

    doi: 10.18632/oncotarget.13137

    Figure Lengend Snippet: Inhibition of miR-29c significantly elevates the migration and invasion ( A ) The expression of mir-29c and Sp1 in TGF-β1-treated cells. Transwell assay was performed and ( B – C ) the migration and invasion of 95C and A549 cells transfected with miR-29c inhibitors were determined. ( D ) The evaluation of transfection efficiency of miR-29c inhibitors. * p

    Article Snippet: E-Cadherin, TIF-1, vimentin, a-SMA and sp1 antibodies were obtained from Cell Signaling Tech (Denver, MA).

    Techniques: Inhibition, Migration, Expressing, Transwell Assay, Transfection

    Sp1 could restore the miR-29c-induced inhibition of metastasis ( A – B ) Overexpression of Sp1 by transfection of pcDNA-Sp1 into 95C and A549 cells. Transwell assay was performed and ( C – D ) the migration and invasion of 95C and A549 cells transfected with miR-29c mimics or pcDNA-Sp1 were determined.

    Journal: Oncotarget

    Article Title: A regulatory loop involving miR-29c and Sp1 elevates the TGF-β1 mediated epithelial-to-mesenchymal transition in lung cancer

    doi: 10.18632/oncotarget.13137

    Figure Lengend Snippet: Sp1 could restore the miR-29c-induced inhibition of metastasis ( A – B ) Overexpression of Sp1 by transfection of pcDNA-Sp1 into 95C and A549 cells. Transwell assay was performed and ( C – D ) the migration and invasion of 95C and A549 cells transfected with miR-29c mimics or pcDNA-Sp1 were determined.

    Article Snippet: E-Cadherin, TIF-1, vimentin, a-SMA and sp1 antibodies were obtained from Cell Signaling Tech (Denver, MA).

    Techniques: Inhibition, Over Expression, Transfection, Transwell Assay, Migration

    MiR-29c could inhibit Sp1/TGF-β1 expression and lung cancer progression ( A ) The TGF-β1 expression in overexpression of miR-29c mimics or negative control was analyzed by Q-PCR in 95C cells with or without the treatment of TGF-β1. ( B – C ) The TGF-β1 expression in overexpression of siRNA-Sp1or negative control was analyzed by Q-PCR in 95C cells with or without the treatment of TGF-β1. ( D ) The relative luciferase activities in 95C cells were determined after the pGL-3-TGFB1 plasmids were transfected with miR-29c mimics. ( E – F ) The relative luciferase activities in 95C cells were determined after the pGL-3-TGFB1 plasmids were transfected with siRNA-Sp1 or pcDNA-Sp1. ( G ) The relative luciferase activities in 95C cells were determined after the pGL-3-TGFB1 plasmids were transfected with pcDNA-Sp1 and miR-29c mimics. ( H – I ) Nude mice (6 per group) were subcutaneously injected of 3 × 10 6 A549 cells and the miR-29c mimics, inhibitor or negative control (10 nM per injection) were delivered via intra-tumoral injection for six times, three days apart. The tumor volume (mm 3 ) and body weight (g) were measured. ( J – K ) The levelS of Sp1 and TGF-β1 in tumor tissues were estimated. * p

    Journal: Oncotarget

    Article Title: A regulatory loop involving miR-29c and Sp1 elevates the TGF-β1 mediated epithelial-to-mesenchymal transition in lung cancer

    doi: 10.18632/oncotarget.13137

    Figure Lengend Snippet: MiR-29c could inhibit Sp1/TGF-β1 expression and lung cancer progression ( A ) The TGF-β1 expression in overexpression of miR-29c mimics or negative control was analyzed by Q-PCR in 95C cells with or without the treatment of TGF-β1. ( B – C ) The TGF-β1 expression in overexpression of siRNA-Sp1or negative control was analyzed by Q-PCR in 95C cells with or without the treatment of TGF-β1. ( D ) The relative luciferase activities in 95C cells were determined after the pGL-3-TGFB1 plasmids were transfected with miR-29c mimics. ( E – F ) The relative luciferase activities in 95C cells were determined after the pGL-3-TGFB1 plasmids were transfected with siRNA-Sp1 or pcDNA-Sp1. ( G ) The relative luciferase activities in 95C cells were determined after the pGL-3-TGFB1 plasmids were transfected with pcDNA-Sp1 and miR-29c mimics. ( H – I ) Nude mice (6 per group) were subcutaneously injected of 3 × 10 6 A549 cells and the miR-29c mimics, inhibitor or negative control (10 nM per injection) were delivered via intra-tumoral injection for six times, three days apart. The tumor volume (mm 3 ) and body weight (g) were measured. ( J – K ) The levelS of Sp1 and TGF-β1 in tumor tissues were estimated. * p

    Article Snippet: E-Cadherin, TIF-1, vimentin, a-SMA and sp1 antibodies were obtained from Cell Signaling Tech (Denver, MA).

    Techniques: Expressing, Over Expression, Negative Control, Polymerase Chain Reaction, Luciferase, Transfection, Mouse Assay, Injection

    MiR-29c targets Sp1 and is down-regulated in high-metastatic lung cancer cell lines ( A – B ) The level of miR-29c and Sp1 in lung cancer tissues and ( C – D ) cell lines including BEAS-2B, the paired low-metastatic 95C and high-metastatic 95D and A549 were determined by Q-PCR and Western blotting. 95C cell line was transfected with miR-29c mimics or negative control. ( E – F ) the mRNA and protein level of Sp1 were determined. ( G – H ) The luciferase reporter was performed to confirm the direct target sites. ** p

    Journal: Oncotarget

    Article Title: A regulatory loop involving miR-29c and Sp1 elevates the TGF-β1 mediated epithelial-to-mesenchymal transition in lung cancer

    doi: 10.18632/oncotarget.13137

    Figure Lengend Snippet: MiR-29c targets Sp1 and is down-regulated in high-metastatic lung cancer cell lines ( A – B ) The level of miR-29c and Sp1 in lung cancer tissues and ( C – D ) cell lines including BEAS-2B, the paired low-metastatic 95C and high-metastatic 95D and A549 were determined by Q-PCR and Western blotting. 95C cell line was transfected with miR-29c mimics or negative control. ( E – F ) the mRNA and protein level of Sp1 were determined. ( G – H ) The luciferase reporter was performed to confirm the direct target sites. ** p

    Article Snippet: E-Cadherin, TIF-1, vimentin, a-SMA and sp1 antibodies were obtained from Cell Signaling Tech (Denver, MA).

    Techniques: Polymerase Chain Reaction, Western Blot, Transfection, Negative Control, Luciferase

    Nilotinib administration induces leukemia regression in mice. A , Approximately 0.1×10 6 C1498 cells were injected into C57BL/6 mice through the tail vein. When the white blood cell counts showed the illness, the leukemic mice were treated with vehicle or nilotinib for 3 weeks. External view (left) and quantification of spleen weight (right) from leukemia-bearing mice are shown. Data are presented as mean values ± S.D. B , Photographs (left) are representative external views of livers from leukemia-bearing mice and the graph (right) shows the quantification of liver weight. Data are shown as mean values ± S.D. C , Pictured are images of lung from leukemia-bearing mice. D , Representative images of H E stained sections of lungs, livers and spleens from leukemia-bearing mice are shown (magnification × 200). E , Wright-Giemsa stained BM cells from leukemia-bearing mice are shown (magnification × 400). F , qPCR was used to determine the expression of DNMT1 , DNMT3a , DNMT3b and Sp1 in BM cells from leukemia-bearing mice. G , The genomic DNA in BM cells from leukemia-bearing mice was isolated and subjected to dotblot analysis. H , qPCR was used to determine the expression of p15 INK4B in BM cells from leukemia-bearing mice. Graph shows the quantification of dot intensities. Note, n = 3 mice/group; data are presented as mean values ± S.D; * P

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Inactivation of receptor tyrosine kinases reverts aberrant DNA methylation in acute myeloid leukemia

    doi: 10.1158/1078-0432.CCR-17-0235

    Figure Lengend Snippet: Nilotinib administration induces leukemia regression in mice. A , Approximately 0.1×10 6 C1498 cells were injected into C57BL/6 mice through the tail vein. When the white blood cell counts showed the illness, the leukemic mice were treated with vehicle or nilotinib for 3 weeks. External view (left) and quantification of spleen weight (right) from leukemia-bearing mice are shown. Data are presented as mean values ± S.D. B , Photographs (left) are representative external views of livers from leukemia-bearing mice and the graph (right) shows the quantification of liver weight. Data are shown as mean values ± S.D. C , Pictured are images of lung from leukemia-bearing mice. D , Representative images of H E stained sections of lungs, livers and spleens from leukemia-bearing mice are shown (magnification × 200). E , Wright-Giemsa stained BM cells from leukemia-bearing mice are shown (magnification × 400). F , qPCR was used to determine the expression of DNMT1 , DNMT3a , DNMT3b and Sp1 in BM cells from leukemia-bearing mice. G , The genomic DNA in BM cells from leukemia-bearing mice was isolated and subjected to dotblot analysis. H , qPCR was used to determine the expression of p15 INK4B in BM cells from leukemia-bearing mice. Graph shows the quantification of dot intensities. Note, n = 3 mice/group; data are presented as mean values ± S.D; * P

    Article Snippet: The antibodies were obtained from Santa Cruz Biotechnology: DNMT3a (sc-20703), Sp1 (sc-59), Bid (sc-11423), Noxa (sc-30209), Bcl-xL (sc-7195) and β-actin (sc-1616); Cell Signaling Technology: PCNA (2586S), phospho-KIT (Tyr719, 3391L), phospho-FLT3 (Tyr589/591, 3464S), phospho-STAT5 (Tyr694, 9351S), phospho-AKT (9272), total KIT (3392S), total FLT3 (3462S) and total STAT5 (9352); New England Biolabs: DNMT1 (M0231L); and Abcam: total AKT (ab126811) and DNMT3b (ab16049).

    Techniques: Mouse Assay, Injection, Staining, Real-time Polymerase Chain Reaction, Expressing, Isolation

    Sp1 activity accounts for the suppression of DNMT1 expression in AML cell lines treated with nilotinib. A-C , Kasumi-1 or MV4-11 cells were treated for 24 h with the indicated doses of nilotinib and subjected to qPCR or Western blot analysis to detect the expression levels of DNMT1, DNMT3a, DNMT3b ( A , B ) and Sp1 ( C ). D and E , EMSA assays were used to detect Sp1 binding to the DNMT1 promoter in Kasumi-1 ( D ) or MV4-11 ( E ) cells treated for 24 h with nilotinib. In D a vertical line was inserted to indicate a repositioned gel lane. F and G , Reporter gene assays were used to determine DNMT1 gene promoter activity in 293T cells transfected with the DNMT1 promoter-luciferase plasmids together with the Sp1 expression vectors ( F ) or treated for 24 h with nilotinib ( G ). H , Western blot analysis of Kasumi-1 and MV4-11 cells transfected for 48 h with Sp1 siRNA (si-) or scramble control. I , Kasumi-1 and MV4-11 cells were transfected with Sp1 expression or control vectors for 12 h and then treated with 10 μM nilotinib for another 24 h. Gene expression was assessed by Western blot analysis. Data represent 3 independent experiments.

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Inactivation of receptor tyrosine kinases reverts aberrant DNA methylation in acute myeloid leukemia

    doi: 10.1158/1078-0432.CCR-17-0235

    Figure Lengend Snippet: Sp1 activity accounts for the suppression of DNMT1 expression in AML cell lines treated with nilotinib. A-C , Kasumi-1 or MV4-11 cells were treated for 24 h with the indicated doses of nilotinib and subjected to qPCR or Western blot analysis to detect the expression levels of DNMT1, DNMT3a, DNMT3b ( A , B ) and Sp1 ( C ). D and E , EMSA assays were used to detect Sp1 binding to the DNMT1 promoter in Kasumi-1 ( D ) or MV4-11 ( E ) cells treated for 24 h with nilotinib. In D a vertical line was inserted to indicate a repositioned gel lane. F and G , Reporter gene assays were used to determine DNMT1 gene promoter activity in 293T cells transfected with the DNMT1 promoter-luciferase plasmids together with the Sp1 expression vectors ( F ) or treated for 24 h with nilotinib ( G ). H , Western blot analysis of Kasumi-1 and MV4-11 cells transfected for 48 h with Sp1 siRNA (si-) or scramble control. I , Kasumi-1 and MV4-11 cells were transfected with Sp1 expression or control vectors for 12 h and then treated with 10 μM nilotinib for another 24 h. Gene expression was assessed by Western blot analysis. Data represent 3 independent experiments.

    Article Snippet: The antibodies were obtained from Santa Cruz Biotechnology: DNMT3a (sc-20703), Sp1 (sc-59), Bid (sc-11423), Noxa (sc-30209), Bcl-xL (sc-7195) and β-actin (sc-1616); Cell Signaling Technology: PCNA (2586S), phospho-KIT (Tyr719, 3391L), phospho-FLT3 (Tyr589/591, 3464S), phospho-STAT5 (Tyr694, 9351S), phospho-AKT (9272), total KIT (3392S), total FLT3 (3462S) and total STAT5 (9352); New England Biolabs: DNMT1 (M0231L); and Abcam: total AKT (ab126811) and DNMT3b (ab16049).

    Techniques: Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Binding Assay, Transfection, Luciferase

    Nilotinib induces global and gene promoter DNA hypomethylation. A , Dotblot analysis using anti-5mC to evaluate changes in global DNA methylation in Kasumi-1 or MV4-11 cells treated for 24 h with nilotinib. B and C , Kasumi-1 or MV4-11 cells were transfected for 48 h with DNMT1 ( B ) or Sp1 siRNA ( C ) or scrambled control. The protein expression levels were assessed by Western blot (upper) and the levels of global DNA methylation were determined by dotblot analysis (lower). D , Kasumi-1 cells were transfected for 12 h with DNMT1 or Sp1 siRNA or scrambled control and then treated with 10 μM nilotinib for another 24 h. Global DNA methylation was determined by dotblot analysis. E , qPCR analysis of p15 INK4B expression in Kasumi-1 or MV4-11 cells treated for 48 h with 10 μM nilotinib. Values are shown as a fold change of gene expression normalized to 18S RNA and compared to vehicle. F , Upper: diagram of the p15 INK4B promoter indicating the location of CpG nucleotides; lower: bisulfite sequencing analysis of changes in p15 INK4B promoter methylation (transcription start site +147 to +221) in Kasumi-1 or MV4-11 cells treated for 48 h with 10 μM nilotinib. Results of 10 clones are presented. Methylated CpG sites are shown as solid circles and open circles indicate non-methylated CpG sites. In A-D , the graphs show the quantification of dot intensity as mean values ± S.D. from 3 independent experiments; * P

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Inactivation of receptor tyrosine kinases reverts aberrant DNA methylation in acute myeloid leukemia

    doi: 10.1158/1078-0432.CCR-17-0235

    Figure Lengend Snippet: Nilotinib induces global and gene promoter DNA hypomethylation. A , Dotblot analysis using anti-5mC to evaluate changes in global DNA methylation in Kasumi-1 or MV4-11 cells treated for 24 h with nilotinib. B and C , Kasumi-1 or MV4-11 cells were transfected for 48 h with DNMT1 ( B ) or Sp1 siRNA ( C ) or scrambled control. The protein expression levels were assessed by Western blot (upper) and the levels of global DNA methylation were determined by dotblot analysis (lower). D , Kasumi-1 cells were transfected for 12 h with DNMT1 or Sp1 siRNA or scrambled control and then treated with 10 μM nilotinib for another 24 h. Global DNA methylation was determined by dotblot analysis. E , qPCR analysis of p15 INK4B expression in Kasumi-1 or MV4-11 cells treated for 48 h with 10 μM nilotinib. Values are shown as a fold change of gene expression normalized to 18S RNA and compared to vehicle. F , Upper: diagram of the p15 INK4B promoter indicating the location of CpG nucleotides; lower: bisulfite sequencing analysis of changes in p15 INK4B promoter methylation (transcription start site +147 to +221) in Kasumi-1 or MV4-11 cells treated for 48 h with 10 μM nilotinib. Results of 10 clones are presented. Methylated CpG sites are shown as solid circles and open circles indicate non-methylated CpG sites. In A-D , the graphs show the quantification of dot intensity as mean values ± S.D. from 3 independent experiments; * P

    Article Snippet: The antibodies were obtained from Santa Cruz Biotechnology: DNMT3a (sc-20703), Sp1 (sc-59), Bid (sc-11423), Noxa (sc-30209), Bcl-xL (sc-7195) and β-actin (sc-1616); Cell Signaling Technology: PCNA (2586S), phospho-KIT (Tyr719, 3391L), phospho-FLT3 (Tyr589/591, 3464S), phospho-STAT5 (Tyr694, 9351S), phospho-AKT (9272), total KIT (3392S), total FLT3 (3462S) and total STAT5 (9352); New England Biolabs: DNMT1 (M0231L); and Abcam: total AKT (ab126811) and DNMT3b (ab16049).

    Techniques: DNA Methylation Assay, Transfection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Methylation Sequencing, Methylation, Clone Assay

    TGFβ signaling modulates c-myc and p21 expression in OP cells through the cooperation of SMAD3/SMAD4 with FoxO1 and Sp1. A , FACS-sorted OPs ( CNP -EGFP low ) were cultured in the presence of TGFβ1 or Vehicle (VEH, control). B , Real-time PCR analysis for the indicated genes from cultured OPs (CNP-EGFP low ) stimulated for 8 h with TGFβ1. C , Luciferase reporter assay activity for the indicated genes was performed in CNP -EGFP low FACS-sorted cells. D–F , Immunofluorescence analysis of OP cultures after 3 h of TGFβ1 treatment shows that TGFβ signaling activation in OPs induces high levels and nuclear localization of pSMAD2/3 ( D , F ), SMAD4, FoxO1 ( E ), and Sp1 ( F ). G , H , Coimmunoprecipitation from OP cell cultures treated with TGFβ1 or vehicle for the indicated antibodies. I , J , ChIP analysis after 4 h of TGFβ1 stimulation of OP cultures. I , Diagram representing the putative binding sites of all TFs analyzed to the promoter region of p21 and c-myc genes. Immunoprecipitation was performed with anti-SMAD3, anti-SMAD4, anti-Sp1, and anti-FoxO1 antibodies or a nonspecific rabbit IgG as a control. J , qPCR analysis revealed increased binding of these four TFs to the promoter region of c-myc and p21 , in TGFβ1-stimulated cells. Results are expressed as arbitrary units after normalization. Data are shown as mean ± SEM; n = 5 brains for each time point. * p

    Journal: The Journal of Neuroscience

    Article Title: TGFβ Signaling Regulates the Timing of CNS Myelination by Modulating Oligodendrocyte Progenitor Cell Cycle Exit through SMAD3/4/FoxO1/Sp1

    doi: 10.1523/JNEUROSCI.0363-14.2014

    Figure Lengend Snippet: TGFβ signaling modulates c-myc and p21 expression in OP cells through the cooperation of SMAD3/SMAD4 with FoxO1 and Sp1. A , FACS-sorted OPs ( CNP -EGFP low ) were cultured in the presence of TGFβ1 or Vehicle (VEH, control). B , Real-time PCR analysis for the indicated genes from cultured OPs (CNP-EGFP low ) stimulated for 8 h with TGFβ1. C , Luciferase reporter assay activity for the indicated genes was performed in CNP -EGFP low FACS-sorted cells. D–F , Immunofluorescence analysis of OP cultures after 3 h of TGFβ1 treatment shows that TGFβ signaling activation in OPs induces high levels and nuclear localization of pSMAD2/3 ( D , F ), SMAD4, FoxO1 ( E ), and Sp1 ( F ). G , H , Coimmunoprecipitation from OP cell cultures treated with TGFβ1 or vehicle for the indicated antibodies. I , J , ChIP analysis after 4 h of TGFβ1 stimulation of OP cultures. I , Diagram representing the putative binding sites of all TFs analyzed to the promoter region of p21 and c-myc genes. Immunoprecipitation was performed with anti-SMAD3, anti-SMAD4, anti-Sp1, and anti-FoxO1 antibodies or a nonspecific rabbit IgG as a control. J , qPCR analysis revealed increased binding of these four TFs to the promoter region of c-myc and p21 , in TGFβ1-stimulated cells. Results are expressed as arbitrary units after normalization. Data are shown as mean ± SEM; n = 5 brains for each time point. * p

    Article Snippet: Immunoprecipitation was performed with anti-SMAD3, anti-SMAD4 (Santa Cruz Biotechnology), anti-Sp1, and anti-FoxO1 (Cell Signaling Technology) antibodies or a nonspecific rabbit IgG as control.

    Techniques: Expressing, FACS, Cell Culture, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay, Activity Assay, Immunofluorescence, Activation Assay, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation

    TGFβ signaling modulates c-myc and p21 expression in OP cells during SCWM. A , G , I , Time line representing the paradigm used for TGFβ1 or vehicle treatment in vivo . B , Immunofluorescence analysis in the SCWM was performed after 4 h of TGFβ1 administration. Activation of TGFβ signaling induces nuclear localization of pSMAD2/3 ( B , C ), SMAD4 ( C , D ), and FoxO1 ( D ) in OPs. E , F , Coimmunoprecipitation analysis of SCWM extracts from TGFβ1 or vehicle (VEH)-treated mice, at 4 h after treatment. TGFβ signaling activation increased the interaction of SMAD3 and SMAD4 with FoxO1 and Sp1 in OPs in the SCWM. SCWM immune complexes were incubated with antibodies against SMAD4 ( E ) or FoxO1 ( F ). As a loading control, coimmunoprecipitation samples were blotted with the same antibodies used to perform the immunoprecipitation. G , H , P5 CNP -EGFP pups received a single administration of Veh or TGFβ1 (100 ng/kg) and 8 h later, CNP -EGFP low (OPs) were FACS sorted to isolate RNA and perform RT-PCR analysis for genes involved in the TGFβ-mediated anti-mitotic program and OL differentiation. I , J , Proteins involved in the TGFβ-mediated anti-mitotic program and OL differentiation were analyzed by WB analysis from total extracts of SCWM after 16 h of treatment. K–M , P5 CNP -EGFP mice received TGFβ1 or vehicle, and immunohistochemistry analysis was performed in the SCWM after 16 h of treatment. Representative confocal images of proteins involved in the cell cycle in OPs ( CNP -EGFP + NG2 + cells), including c-myc ( K ), p21 ( L ), and p27 ( M ) after 16 h of TGFβ1 treatment. Histograms express results in a.u. after normalization. n = 5 brains for each time point. ** p

    Journal: The Journal of Neuroscience

    Article Title: TGFβ Signaling Regulates the Timing of CNS Myelination by Modulating Oligodendrocyte Progenitor Cell Cycle Exit through SMAD3/4/FoxO1/Sp1

    doi: 10.1523/JNEUROSCI.0363-14.2014

    Figure Lengend Snippet: TGFβ signaling modulates c-myc and p21 expression in OP cells during SCWM. A , G , I , Time line representing the paradigm used for TGFβ1 or vehicle treatment in vivo . B , Immunofluorescence analysis in the SCWM was performed after 4 h of TGFβ1 administration. Activation of TGFβ signaling induces nuclear localization of pSMAD2/3 ( B , C ), SMAD4 ( C , D ), and FoxO1 ( D ) in OPs. E , F , Coimmunoprecipitation analysis of SCWM extracts from TGFβ1 or vehicle (VEH)-treated mice, at 4 h after treatment. TGFβ signaling activation increased the interaction of SMAD3 and SMAD4 with FoxO1 and Sp1 in OPs in the SCWM. SCWM immune complexes were incubated with antibodies against SMAD4 ( E ) or FoxO1 ( F ). As a loading control, coimmunoprecipitation samples were blotted with the same antibodies used to perform the immunoprecipitation. G , H , P5 CNP -EGFP pups received a single administration of Veh or TGFβ1 (100 ng/kg) and 8 h later, CNP -EGFP low (OPs) were FACS sorted to isolate RNA and perform RT-PCR analysis for genes involved in the TGFβ-mediated anti-mitotic program and OL differentiation. I , J , Proteins involved in the TGFβ-mediated anti-mitotic program and OL differentiation were analyzed by WB analysis from total extracts of SCWM after 16 h of treatment. K–M , P5 CNP -EGFP mice received TGFβ1 or vehicle, and immunohistochemistry analysis was performed in the SCWM after 16 h of treatment. Representative confocal images of proteins involved in the cell cycle in OPs ( CNP -EGFP + NG2 + cells), including c-myc ( K ), p21 ( L ), and p27 ( M ) after 16 h of TGFβ1 treatment. Histograms express results in a.u. after normalization. n = 5 brains for each time point. ** p

    Article Snippet: Immunoprecipitation was performed with anti-SMAD3, anti-SMAD4 (Santa Cruz Biotechnology), anti-Sp1, and anti-FoxO1 (Cell Signaling Technology) antibodies or a nonspecific rabbit IgG as control.

    Techniques: Expressing, In Vivo, Immunofluorescence, Activation Assay, Mouse Assay, Incubation, Immunoprecipitation, FACS, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunohistochemistry

    Proposed model of TGFβ signaling modulation of oligodendrogenesis during the SCWM myelination. TGFβ signaling in OPs controls the canonical downstream TGFβ-R effectors, SMAD2/3/4. Upon TGFβ-R's activation, the heteromeric SMAD2/3/4 complex establishes a nuclear localization, and cooperates with FoxO1 and Sp1 to modulate the transcription of c-myc and p21. Upon binding regulatory sequences, SMAD2/3/4, FoxO1, and Sp1 cooperate to repress the transcription of c-myc and activate p21 transcription, allowing OPs to withdraw from the cell cycle and progress down OL differentiation and maturation.

    Journal: The Journal of Neuroscience

    Article Title: TGFβ Signaling Regulates the Timing of CNS Myelination by Modulating Oligodendrocyte Progenitor Cell Cycle Exit through SMAD3/4/FoxO1/Sp1

    doi: 10.1523/JNEUROSCI.0363-14.2014

    Figure Lengend Snippet: Proposed model of TGFβ signaling modulation of oligodendrogenesis during the SCWM myelination. TGFβ signaling in OPs controls the canonical downstream TGFβ-R effectors, SMAD2/3/4. Upon TGFβ-R's activation, the heteromeric SMAD2/3/4 complex establishes a nuclear localization, and cooperates with FoxO1 and Sp1 to modulate the transcription of c-myc and p21. Upon binding regulatory sequences, SMAD2/3/4, FoxO1, and Sp1 cooperate to repress the transcription of c-myc and activate p21 transcription, allowing OPs to withdraw from the cell cycle and progress down OL differentiation and maturation.

    Article Snippet: Immunoprecipitation was performed with anti-SMAD3, anti-SMAD4 (Santa Cruz Biotechnology), anti-Sp1, and anti-FoxO1 (Cell Signaling Technology) antibodies or a nonspecific rabbit IgG as control.

    Techniques: Activation Assay, Binding Assay

    EMSA of (A) I Sp1 (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.

    Journal: PLoS ONE

    Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

    doi: 10.1371/journal.pone.0005049

    Figure Lengend Snippet: EMSA of (A) I Sp1 (−496), (B) II Sp1 (−303), (C) III Sp1 (−114), (D) NF-κB (−354). Lane 1, biotin-labeled oligonucleotide alone; lane 2, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts; lane 3, biotin-labeled oligonucleotides incubated with 5 μg hyperosmolarity-treated Caco2-BBE nuclear extracts; lane 4, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of anti-Sp1 (A–C) or NF-kB (p65) (D) antibodies; lane 5, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of non-specific IgG; lane 6, biotin-labeled oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts in the presence of a 50-fold excess of cold competitor oligonucleotide; lane 7, biotin-labeled binding site-mutated oligonucleotides incubated with 5 μg Caco2-BBE nuclear extracts. E. Chromatin immunoprecipitation (ChIP) assay: the antibodies indicated were incubated with cross-linked DNA isolated from Caco2-BBE cells treated with (+) or without (−) hyperosmolarity, IgG antisera acts as control. Sp1 (I, II, and III) and NF-κB promoter elements in the immunoprecipitates were detected by PCR. The lower panel shows DNA input as template for internal control.

    Article Snippet: Samples were then immunoprecipitated with 2 μg of mouse anti-Sp1 (Upstate Cell Signaling Solutions) or 3 μg of rabbit anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Labeling, Incubation, Binding Assay, Chromatin Immunoprecipitation, Isolation, Polymerase Chain Reaction

    Characterization of SPAK promoter. A. Schematic representation of human SPAK promoter constructs. the full-length SPAK promoter (nt-1472 to +4); construct I (nt −1050 to +4); construct II (nt −398 to +4); construct III (nt −331 to +4); construct IV (nt −149 to +4) and construct V (nt −72 to +4). Numbers are given in relation to the translational start codon (+1) and indicate 5′-ends of the deletion constructs. The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 (Red) and NF-κB (Yellow) sites are indicated by arrows. B. Promoter activities of the 5′ deleted constructs in un-treated or hyperosmolarity-stimulated Caco2-BBE cells normalized to Renilla Luc activities driven by the phRL-CMV control vector. Activities are expressed as fold inductions over cells transfected with the empty pGL3-basic vector. Each value represents the mean±SD of at least 3 independent sets of transfection experiments performed in triplicate, *p

    Journal: PLoS ONE

    Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

    doi: 10.1371/journal.pone.0005049

    Figure Lengend Snippet: Characterization of SPAK promoter. A. Schematic representation of human SPAK promoter constructs. the full-length SPAK promoter (nt-1472 to +4); construct I (nt −1050 to +4); construct II (nt −398 to +4); construct III (nt −331 to +4); construct IV (nt −149 to +4) and construct V (nt −72 to +4). Numbers are given in relation to the translational start codon (+1) and indicate 5′-ends of the deletion constructs. The location of the identified positive regulatory region is indicated by a light blue box. Positions of the putative Sp1 (Red) and NF-κB (Yellow) sites are indicated by arrows. B. Promoter activities of the 5′ deleted constructs in un-treated or hyperosmolarity-stimulated Caco2-BBE cells normalized to Renilla Luc activities driven by the phRL-CMV control vector. Activities are expressed as fold inductions over cells transfected with the empty pGL3-basic vector. Each value represents the mean±SD of at least 3 independent sets of transfection experiments performed in triplicate, *p

    Article Snippet: Samples were then immunoprecipitated with 2 μg of mouse anti-Sp1 (Upstate Cell Signaling Solutions) or 3 μg of rabbit anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Construct, Plasmid Preparation, Transfection

    Western blots of transcription factors Sp1 and NF-κB (p65). A. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vivo . Histone3 acts as a control. B. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vitro . Histone3 acts as a control. C. Reduction of NF-κB but not Sp1 expression reduced SPAK protein expression in unstimulated and in hyperosmolarity-stimulated Caco2-BBE cells. Cells were harvested and subjected to western blot analysis using Sp1, NF-κB (p65), and SPAK antibodies as described in materials and methods . GAPDH acts as a loading control.

    Journal: PLoS ONE

    Article Title: Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

    doi: 10.1371/journal.pone.0005049

    Figure Lengend Snippet: Western blots of transcription factors Sp1 and NF-κB (p65). A. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vivo . Histone3 acts as a control. B. Western blots of Sp1 and NF-κB (p65) demonstrating hyperosmolarity effect on Sp1 and NF-κB protein levels in vitro . Histone3 acts as a control. C. Reduction of NF-κB but not Sp1 expression reduced SPAK protein expression in unstimulated and in hyperosmolarity-stimulated Caco2-BBE cells. Cells were harvested and subjected to western blot analysis using Sp1, NF-κB (p65), and SPAK antibodies as described in materials and methods . GAPDH acts as a loading control.

    Article Snippet: Samples were then immunoprecipitated with 2 μg of mouse anti-Sp1 (Upstate Cell Signaling Solutions) or 3 μg of rabbit anti-p65 antibody (Santa Cruz Biotechnology) overnight at 4°C.

    Techniques: Western Blot, In Vivo, In Vitro, Expressing