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cdk9 d 7  (Santa Cruz Biotechnology)


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    Structured Review

    Santa Cruz Biotechnology cdk9 d 7
    (A) Neurons were incubated for 3 h with ACBI (2.5 μM), and activity was induced by Bic + 4AP for 15 min. CoIP was then performed in neuronal lysates with <t>anti-CDK9.</t> CoIP samples were separated on a gel and blotted for other BAF subunits. SMARCA4 is used as a control to show ACBI efficiency. Spt6 is part of the elongation complex. (B) Neurons were incubated with MC180 or Thal-SNS-032 for 20 min or 3 h, respectively, followed by activity induction for 15 min with Bic + 4AP. Normalized Arc pre-mRNA levels were assayed and are displayed. (C) Neurons were treated as indicated and fractionated. The chromatin fraction was used, and anti-SMARCC2 coIP samples were electrophoresed and blotted for other BAF subunits. ARID1A is shown on a blot separate from the others. The ARID1A loading control (SMARCC2) is not displayed here to avoid duplication but is quantified in (D). (D and E) Quantification of (C) for ARID1A and SS18L1, respectively (normalized by SMARCC2). Quantification was performed as described for . (F) ChIP data to show SMARCC2 binding inside the Arc gene body 15 min after stimulation of wild type or neurons depleted of the subunit. (G) Quantified elongating RNA Pol II binding inside the Arc gene body, 15 min after stimulation, determined by ChIP with antibody against Rpb1-pSer2. (H) Quantified SMARCC2 binding inside the Arc gene body, after identical stimulation as in (G), determined by ChIP with antibody against SMARCC2. Gray dots represent biological replicates; error bars show SE of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA was performed followed by Tukey’s post hoc test.
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    Images

    1) Product Images from "Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation"

    Article Title: Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation

    Journal: Cell reports

    doi: 10.1016/j.celrep.2024.114877

    (A) Neurons were incubated for 3 h with ACBI (2.5 μM), and activity was induced by Bic + 4AP for 15 min. CoIP was then performed in neuronal lysates with anti-CDK9. CoIP samples were separated on a gel and blotted for other BAF subunits. SMARCA4 is used as a control to show ACBI efficiency. Spt6 is part of the elongation complex. (B) Neurons were incubated with MC180 or Thal-SNS-032 for 20 min or 3 h, respectively, followed by activity induction for 15 min with Bic + 4AP. Normalized Arc pre-mRNA levels were assayed and are displayed. (C) Neurons were treated as indicated and fractionated. The chromatin fraction was used, and anti-SMARCC2 coIP samples were electrophoresed and blotted for other BAF subunits. ARID1A is shown on a blot separate from the others. The ARID1A loading control (SMARCC2) is not displayed here to avoid duplication but is quantified in (D). (D and E) Quantification of (C) for ARID1A and SS18L1, respectively (normalized by SMARCC2). Quantification was performed as described for . (F) ChIP data to show SMARCC2 binding inside the Arc gene body 15 min after stimulation of wild type or neurons depleted of the subunit. (G) Quantified elongating RNA Pol II binding inside the Arc gene body, 15 min after stimulation, determined by ChIP with antibody against Rpb1-pSer2. (H) Quantified SMARCC2 binding inside the Arc gene body, after identical stimulation as in (G), determined by ChIP with antibody against SMARCC2. Gray dots represent biological replicates; error bars show SE of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA was performed followed by Tukey’s post hoc test.
    Figure Legend Snippet: (A) Neurons were incubated for 3 h with ACBI (2.5 μM), and activity was induced by Bic + 4AP for 15 min. CoIP was then performed in neuronal lysates with anti-CDK9. CoIP samples were separated on a gel and blotted for other BAF subunits. SMARCA4 is used as a control to show ACBI efficiency. Spt6 is part of the elongation complex. (B) Neurons were incubated with MC180 or Thal-SNS-032 for 20 min or 3 h, respectively, followed by activity induction for 15 min with Bic + 4AP. Normalized Arc pre-mRNA levels were assayed and are displayed. (C) Neurons were treated as indicated and fractionated. The chromatin fraction was used, and anti-SMARCC2 coIP samples were electrophoresed and blotted for other BAF subunits. ARID1A is shown on a blot separate from the others. The ARID1A loading control (SMARCC2) is not displayed here to avoid duplication but is quantified in (D). (D and E) Quantification of (C) for ARID1A and SS18L1, respectively (normalized by SMARCC2). Quantification was performed as described for . (F) ChIP data to show SMARCC2 binding inside the Arc gene body 15 min after stimulation of wild type or neurons depleted of the subunit. (G) Quantified elongating RNA Pol II binding inside the Arc gene body, 15 min after stimulation, determined by ChIP with antibody against Rpb1-pSer2. (H) Quantified SMARCC2 binding inside the Arc gene body, after identical stimulation as in (G), determined by ChIP with antibody against SMARCC2. Gray dots represent biological replicates; error bars show SE of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA was performed followed by Tukey’s post hoc test.

    Techniques Used: Incubation, Activity Assay, Control, Binding Assay

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Plasmid Preparation, Magnetic Beads, One Step RT-PCR, SYBR Green Assay, Mass Spectrometry, Software

    Related Articles

    Bioprocessing:

    Article Title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ
    Article Snippet: Mouse monoclonal anti-FLAG peptide (clone M2) and horseradish peroxidase-conjugated antibodies were purchased from Sigma-Aldrich. .. Mouse monoclonal antibodies against SRPK1, SRPK2 (23/SRPK2), and CDK9 (D-7) were purchased from Pharmingen (BD Biosciences, San Jose, CA, USA), BD Transduction Laboratories (BD Biosciences), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. .. Mouse monoclonal antibody against GAPDH (6C5) and rabbit polyclonal antibodies against CLK1 and Haspin were obtained from Abcam (Cambridge, UK).

    Article Title: Identification of a DYRK1A Inhibitor that Induces Degradation of the Target Kinase using Co-chaperone CDC37 fused with Luciferase nanoKAZ.
    Article Snippet: Mouse monoclonal anti-FLAG peptide (clone M2) and horseradish peroxidase-conjugated antibodies were purchased from Sigma-Aldrich. .. Mouse monoclonal antibodies against SRPK1, SRPK2 (23/ SRPK2), and CDK9 (D-7) were purchased from Pharmingen (BD Biosciences, San Jose, CA, USA), BD Transduction Laboratories (BD Biosciences), and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. .. Mouse monoclonal antibody against GAPDH (6C5) and rabbit polyclonal antibodies against CLK1 and Haspin were obtained from Abcam (Cambridge, UK).

    other:

    Article Title: Nuclear microRNAs release paused Pol II via the DDX21-CDK9 complex.
    Article Snippet: For proteome analysis, beads bound to proteins were digested with LysC/trypsin (Promega) in 50 mM Tris-HCl (pH 8.0), and then incubated at 37 C overnight.

    Article Title: Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation
    Article Snippet: CDK9 (D-7) , Santa Cruz Biotechnology , Cat#: sc-13130 Mouse mAb RRID: AB_627245.

    Co-Immunoprecipitation Assay:

    Article Title: Nuclear microRNAs release paused Pol II via the DDX21-CDK9 complex.
    Article Snippet: Two micrograms of each antibody was bound to 10 mL of SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories) and incubated with the nuclear extract for 1 h at room temperature with or without 100 mg/mL RNase A. .. After one wash with TBS-T, Co-IP complex proteins were detected by immunoblotting using antibodies against AGO1 (D84G10) (Cell Signaling Technology, #5053), AGO2 (Abcam, ab32381), DDX21 (Proteintech, 10528-1-AP), or Cdk9 (D-7) (Santa Cruz, sc-13130). .. For proteome analysis, beads bound to proteins were digested with LysC/trypsin (Promega) in 50 mM Tris-HCl (pH 8.0), and then incubated at 37 C overnight.

    Western Blot:

    Article Title: Nuclear microRNAs release paused Pol II via the DDX21-CDK9 complex.
    Article Snippet: Two micrograms of each antibody was bound to 10 mL of SureBeads Protein G Magnetic Beads (Bio-Rad Laboratories) and incubated with the nuclear extract for 1 h at room temperature with or without 100 mg/mL RNase A. .. After one wash with TBS-T, Co-IP complex proteins were detected by immunoblotting using antibodies against AGO1 (D84G10) (Cell Signaling Technology, #5053), AGO2 (Abcam, ab32381), DDX21 (Proteintech, 10528-1-AP), or Cdk9 (D-7) (Santa Cruz, sc-13130). .. For proteome analysis, beads bound to proteins were digested with LysC/trypsin (Promega) in 50 mM Tris-HCl (pH 8.0), and then incubated at 37 C overnight.



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    (A) Neurons were incubated for 3 h with ACBI (2.5 μM), and activity was induced by Bic + 4AP for 15 min. CoIP was then performed in neuronal lysates with <t>anti-CDK9.</t> CoIP samples were separated on a gel and blotted for other BAF subunits. SMARCA4 is used as a control to show ACBI efficiency. Spt6 is part of the elongation complex. (B) Neurons were incubated with MC180 or Thal-SNS-032 for 20 min or 3 h, respectively, followed by activity induction for 15 min with Bic + 4AP. Normalized Arc pre-mRNA levels were assayed and are displayed. (C) Neurons were treated as indicated and fractionated. The chromatin fraction was used, and anti-SMARCC2 coIP samples were electrophoresed and blotted for other BAF subunits. ARID1A is shown on a blot separate from the others. The ARID1A loading control (SMARCC2) is not displayed here to avoid duplication but is quantified in (D). (D and E) Quantification of (C) for ARID1A and SS18L1, respectively (normalized by SMARCC2). Quantification was performed as described for . (F) ChIP data to show SMARCC2 binding inside the Arc gene body 15 min after stimulation of wild type or neurons depleted of the subunit. (G) Quantified elongating RNA Pol II binding inside the Arc gene body, 15 min after stimulation, determined by ChIP with antibody against Rpb1-pSer2. (H) Quantified SMARCC2 binding inside the Arc gene body, after identical stimulation as in (G), determined by ChIP with antibody against SMARCC2. Gray dots represent biological replicates; error bars show SE of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA was performed followed by Tukey’s post hoc test.
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    Image Search Results


    (A) Neurons were incubated for 3 h with ACBI (2.5 μM), and activity was induced by Bic + 4AP for 15 min. CoIP was then performed in neuronal lysates with anti-CDK9. CoIP samples were separated on a gel and blotted for other BAF subunits. SMARCA4 is used as a control to show ACBI efficiency. Spt6 is part of the elongation complex. (B) Neurons were incubated with MC180 or Thal-SNS-032 for 20 min or 3 h, respectively, followed by activity induction for 15 min with Bic + 4AP. Normalized Arc pre-mRNA levels were assayed and are displayed. (C) Neurons were treated as indicated and fractionated. The chromatin fraction was used, and anti-SMARCC2 coIP samples were electrophoresed and blotted for other BAF subunits. ARID1A is shown on a blot separate from the others. The ARID1A loading control (SMARCC2) is not displayed here to avoid duplication but is quantified in (D). (D and E) Quantification of (C) for ARID1A and SS18L1, respectively (normalized by SMARCC2). Quantification was performed as described for . (F) ChIP data to show SMARCC2 binding inside the Arc gene body 15 min after stimulation of wild type or neurons depleted of the subunit. (G) Quantified elongating RNA Pol II binding inside the Arc gene body, 15 min after stimulation, determined by ChIP with antibody against Rpb1-pSer2. (H) Quantified SMARCC2 binding inside the Arc gene body, after identical stimulation as in (G), determined by ChIP with antibody against SMARCC2. Gray dots represent biological replicates; error bars show SE of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA was performed followed by Tukey’s post hoc test.

    Journal: Cell reports

    Article Title: Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation

    doi: 10.1016/j.celrep.2024.114877

    Figure Lengend Snippet: (A) Neurons were incubated for 3 h with ACBI (2.5 μM), and activity was induced by Bic + 4AP for 15 min. CoIP was then performed in neuronal lysates with anti-CDK9. CoIP samples were separated on a gel and blotted for other BAF subunits. SMARCA4 is used as a control to show ACBI efficiency. Spt6 is part of the elongation complex. (B) Neurons were incubated with MC180 or Thal-SNS-032 for 20 min or 3 h, respectively, followed by activity induction for 15 min with Bic + 4AP. Normalized Arc pre-mRNA levels were assayed and are displayed. (C) Neurons were treated as indicated and fractionated. The chromatin fraction was used, and anti-SMARCC2 coIP samples were electrophoresed and blotted for other BAF subunits. ARID1A is shown on a blot separate from the others. The ARID1A loading control (SMARCC2) is not displayed here to avoid duplication but is quantified in (D). (D and E) Quantification of (C) for ARID1A and SS18L1, respectively (normalized by SMARCC2). Quantification was performed as described for . (F) ChIP data to show SMARCC2 binding inside the Arc gene body 15 min after stimulation of wild type or neurons depleted of the subunit. (G) Quantified elongating RNA Pol II binding inside the Arc gene body, 15 min after stimulation, determined by ChIP with antibody against Rpb1-pSer2. (H) Quantified SMARCC2 binding inside the Arc gene body, after identical stimulation as in (G), determined by ChIP with antibody against SMARCC2. Gray dots represent biological replicates; error bars show SE of the mean. * p < 0.05, ** p < 0.01, and *** p < 0.001. One-way ANOVA was performed followed by Tukey’s post hoc test.

    Article Snippet: CDK9 (D-7) , Santa Cruz Biotechnology , Cat#: sc-13130 Mouse mAb RRID: AB_627245.

    Techniques: Incubation, Activity Assay, Control, Binding Assay

    KEY RESOURCES TABLE

    Journal: Cell reports

    Article Title: Activity-assembled nBAF complex mediates rapid immediate early gene transcription by regulating RNA polymerase II productive elongation

    doi: 10.1016/j.celrep.2024.114877

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: CDK9 (D-7) , Santa Cruz Biotechnology , Cat#: sc-13130 Mouse mAb RRID: AB_627245.

    Techniques: Recombinant, Plasmid Preparation, Magnetic Beads, One Step RT-PCR, SYBR Green Assay, Mass Spectrometry, Software

    Journal: iScience

    Article Title: Evolutionary analysis reveals the role of a non-catalytic domain of peptidyl arginine deiminase 2 in transcriptional regulation

    doi: 10.1016/j.isci.2024.109584

    Figure Lengend Snippet:

    Article Snippet: Rabbit polyclonal anti-CDK9 (D-7) , Santa Cruz Biot. , SC-13130.

    Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Clone Assay, Plasmid Preparation, Mutagenesis, Software