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cdk9 d 7  (Santa Cruz Biotechnology)


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    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



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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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    (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.

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    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.

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