polyclonal rabbit sin3a antibody  (21st Century Biochemicals)

Journal: bioRxiv

Article Title: Distinct regions within SAP25 recruit O-linked glycosylation, DNA demethylation, and ubiquitin ligase and hydrolase activities to the Sin3/HDAC complex

doi: 10.1101/2024.03.05.583553

Figure Lengend Snippet: A. SAP25 region 16-30 is sufficient and region 21-40 is required for interaction with SCF(FBXO3). Quantitative (dNSAF) values for SAP25, SCF(FBXO3) complex subunits, and SIN3A are shown for Halo-SAP25 WT Halo-SAP25 16-30 and Halo-SAP25 Δ21-40purifications. Bait proteins were transiently expressed in 293T cells. Values are the average of at least three biological replicates and error bars represent standard deviation. B. FBXO3captures SAP25 but no other Sin3 complex subunits. SNAP-FLAG-FBXO3 was co-expressed with the indicated Halo-tagged proteins and the resulting lysates subjected to SNAP affinity purification. Copurified proteins were identified by SDS-PAGE followed by Western blotting using the indicated antibodies. C. Amino acid positions within SAP25 region 16-30 are important for FBXO3 capture Proteins captured by either Halo-SAP25or Halo-SAP25 S26/27/40D T35E were resolved by SDS-PAGE and detected by Western blotting using the indicated antibodies. Mutated amino acids within an N-terminal region are highlighted in red. D. Inferred position within SAP25 required for FBXO3 binding. Possible abrogation of SAP25/FBXO3 interaction by modified residues within the binding region are indicated by red Ps.

Article Snippet: Rabbit anti-SAP30 (ab125187) and rabbit anti-SIN3A (ab3479) polyclonal antibodies were from Abcam (Cambridge, United Kingdom).

Techniques: Standard Deviation, Affinity Purification, SDS Page, Western Blot, Binding Assay, Modification

Journal: Journal of Dental Sciences

Article Title: Butyrate-treatment induces gingival epithelial cell death in a three-dimensional gingival-connective tissue hybrid co-culture system

doi: 10.1016/j.jds.2022.08.034

Figure Lengend Snippet: Butyrate treatment induces the release of cellular components from gingival epithelial layers of the 3D gingival culture system. (A) Time-course of the amount of DNA released during butyrate treatment. The amount of DNA in the conditioned media was measured using SYTOX-green dye (n = 4, ∗, P < 0.01 compared to the control treatment). (B) Butyrate treatment for 72 h induces dose-dependent DNA release from epithelial cells of the 3D gingival culture system (n = 4, ∗, P < 0.01 compared to control treatment). (C) Western blot analysis using conditioned media showed that butyrate treatment for 72 h induced SAP130 release from the epithelial layer of the 3D gingival culture system. (D) Time-course of the amount of DNA released during butyrate treatment of the 2D Ca9-22 cell culture (n = 4, ∗, P < 0.01 compared to control treatment).

Article Snippet: Aliquots of cell culture supernatant (20 μL/lane) were subjected to western blotting using an anti-Sin3A associated protein 130 kDa (SAP130) rabbit polyclonal antibody (GeneTex, Irvine, CA, USA) as the primary antibody and WestVision secondary antibody labeled with horseradish peroxidase (Vector Laboratories, Burlingame, CA, USA).

Techniques: Control, Western Blot, Cell Culture

Journal: eLife

Article Title: Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters

doi: 10.7554/eLife.36435

Figure Lengend Snippet: ( A ) Nuclear lysates prepared under three different conditions from E10.5 embryos harboring the Fam60a::Venus transgene were subjected to immunoprecipitation with bead-coupled antibodies to GFP that either had (control) or had not been previously exposed to recombinant GFP. Proteins that bound to the beads were then fractionated by SDS-polyacrylamide gel electrophoresis and revealed by silver staining. Proteins that bound nonspecifically to the beads are indicated by asterisks, and the identity of those that bound specifically was determined by mass spectrometry. The bead-bound proteins were also subjected to immunoblot (IB) analysis with antibodies to GFP for detection of Fam60a-Venus. ( B ) Nuclear extracts of E10.5 WT embryos, undifferentiated P19 cells, or E10.5 embryos harboring the Fam60a::Venus transgene were subjected to immunoprecipitation (IP) with bead-coupled anti-Fam60a, anti-GFP, or control rabbit immunoglobulin G (IgG), as indicated, and the resulting precipitates were subjected to immunoblot analysis with antibodies to Sin3a, to Hdac1, and to Hdac2 (upper panel). The nuclear extracts (Input) as well as the material that bound nonspecifically to beads before exposure to the antibodies used for immunoprecipitation (Preclear) were also subjected to immunoblot analysis. Alternatively, nuclear extracts of E10.5 WT embryos were subjected to immunoprecipitation with antibodies to Sin3a, to Hdac1, to Hdac2, or to the FLAG epitope (control), and the resulting precipitates were subjected to immunoblot analysis with antibodies to Fam60a (lower panel). See also .

Article Snippet: Additional antibodies included control rabbit IgG (Kamiya Biomedical or Thermo Fisher Scientific), conformation-specific mouse monoclonal antibodies to rabbit IgG (#3678, Cell Signaling) that can avoid detection of denatured rabbit IgG used for immunoprecipitation, as well as rabbit polyclonal antibodies to GFP (598, MBL International), to Hdac1 (ab31263, Abcam), to FLAG (F3165, Sigma-Aldrich), to Hdac2 (ab7029, Abcam), to Sin3a (sc-994, Santa Cruz Biotechnology), to Ing2 (ab109504, Abcam), to RbAp46/48 (39199, Active Motif), to AcH3K9 (39917, Active Motif), to BrdU (347580, BD Biosciences), and to 5hmC (39769, Active Motif).

Techniques: Immunoprecipitation, Recombinant, Polyacrylamide Gel Electrophoresis, Silver Staining, Mass Spectrometry, Western Blot, FLAG-tag

Journal: eLife

Article Title: Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters

doi: 10.7554/eLife.36435

Figure Lengend Snippet: Nuclear extracts prepared from Fam60a flox/– and Fam60a –/– ES cells were subjected to immunoprecipitation with antibodies to Sin3a, and the resulting precipitates were fractionated by SDS-polyacrylamide gel electrophoresis and stained with silver (left panel). The gel was also subjected to immunoblot analysis with antibodies to the indicated proteins (right panel).

Article Snippet: Additional antibodies included control rabbit IgG (Kamiya Biomedical or Thermo Fisher Scientific), conformation-specific mouse monoclonal antibodies to rabbit IgG (#3678, Cell Signaling) that can avoid detection of denatured rabbit IgG used for immunoprecipitation, as well as rabbit polyclonal antibodies to GFP (598, MBL International), to Hdac1 (ab31263, Abcam), to FLAG (F3165, Sigma-Aldrich), to Hdac2 (ab7029, Abcam), to Sin3a (sc-994, Santa Cruz Biotechnology), to Ing2 (ab109504, Abcam), to RbAp46/48 (39199, Active Motif), to AcH3K9 (39917, Active Motif), to BrdU (347580, BD Biosciences), and to 5hmC (39769, Active Motif).

Techniques: Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Staining, Western Blot

Journal: eLife

Article Title: Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters

doi: 10.7554/eLife.36435

Figure Lengend Snippet: ( A ) Average binding profile for Fam60a-Venus at the TSS region of all genes with binding peaks identified by ChIP-seq analysis of E9.5 transgenic embryos with antibodies to GFP. Distance is expressed relative to the TSS. ( B ) Peak distribution for ChIP-seq analysis as in ( A ). About 80% of peaks were localized to gene loci. UTR, untranslated region. ( C ) Venn diagram showing the overlap in Fam60a target genes (those with binding peaks within ±3 kb of the TSS) for two independent ChIP-seq analyses (ChIP-seq1 and ChIP-seq2). ( D ) ChIP-qPCR analysis of the binding of Fam60a-Venus and Sin3a to the TSS regions of the indicated genes in E9.5 transgenic and WT embryos, respectively. The pale blue and orange bars represent IgG controls for antibodies to GFP and to Sin3a, respectively. Data are expressed as percentage of input and are means ± s.d. for three independent experiments. Actb was examined as a positive control. ( E ) Examples of Fam60a-Venus ChIP-seq results for E9.5 Fam60a-Venus embryos. ChIP-seq1 and ChIP-seq2 were both performed with antibodies to GFP. Peaks around the TSS are shown for four Fam60a target genes, with red arrows indicating the direction of transcription. See also and . 10.7554/eLife.36435.028 Figure 5—source data 1. List of target genomic regions identified by ChIP-seq analysis. 10.7554/eLife.36435.029 Figure 5—source data 2. Numerical data of . 10.7554/eLife.36435.030 Figure 5—source data 3. Numerical data of .

Article Snippet: Additional antibodies included control rabbit IgG (Kamiya Biomedical or Thermo Fisher Scientific), conformation-specific mouse monoclonal antibodies to rabbit IgG (#3678, Cell Signaling) that can avoid detection of denatured rabbit IgG used for immunoprecipitation, as well as rabbit polyclonal antibodies to GFP (598, MBL International), to Hdac1 (ab31263, Abcam), to FLAG (F3165, Sigma-Aldrich), to Hdac2 (ab7029, Abcam), to Sin3a (sc-994, Santa Cruz Biotechnology), to Ing2 (ab109504, Abcam), to RbAp46/48 (39199, Active Motif), to AcH3K9 (39917, Active Motif), to BrdU (347580, BD Biosciences), and to 5hmC (39769, Active Motif).

Techniques: Binding Assay, ChIP-sequencing, Transgenic Assay, Positive Control

Journal: eLife

Article Title: Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters

doi: 10.7554/eLife.36435

Figure Lengend Snippet: ( A ) Molecular phylogeny of Fam60a and related proteins. The tree was inferred with the maximum-likelihood method and 99 amino acid residues. Bootstrap values are indicated at individual nodes. ( B ) Gene repertories for Fam60, Sin3, Tet, and Dnmt families. Black boxes indicate the presence of at least one phylogenetically validated ortholog, whereas white boxes indicate the absence of orthologs. 2R-WGD, two rounds of whole-genome duplication. The presence or absence of DNA methylation in individual species based on current knowledge ( ; ) is also shown. ( C and D ) Fam60a inhibits Tet1 activity in NIH3T3 cells. Immunofluorescence staining of 5hmC and FLAG-Tet1 was performed for NIH3T3 cells expressing FLAG-Tet1 either together with Fam60a ( C ) or alone ( D ). The cells were analyzed 24 hr after the induction of FLAG-Tet1 expression by doxycycline administration. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). Red arrows indicate cells positive for both 5hmC and FLAG immunoreactivity. White arrows indicate cells positive for FLAG but negative for 5hmC. ( E ) Plots of mean fluorescence intensity for 5hmC versus FLAG-Tet1 in cells expressing FLAG-Tet1 without (left) or with (right) Fam60a as in ( C ) and ( D ). ( F ) Proportion of FLAG-Tet1 + cells that were negative for 5hmC in experiments similar to that in ( C ) and ( D ). Data are means ± s.d. for three independent experiments. *p<0.05 (Student’s unpaired t test). See also and . 10.7554/eLife.36435.034 Figure 6—source data 1. Numerical data of . 10.7554/eLife.36435.035 Figure 6—source data 2. Numerical data of .

Article Snippet: Additional antibodies included control rabbit IgG (Kamiya Biomedical or Thermo Fisher Scientific), conformation-specific mouse monoclonal antibodies to rabbit IgG (#3678, Cell Signaling) that can avoid detection of denatured rabbit IgG used for immunoprecipitation, as well as rabbit polyclonal antibodies to GFP (598, MBL International), to Hdac1 (ab31263, Abcam), to FLAG (F3165, Sigma-Aldrich), to Hdac2 (ab7029, Abcam), to Sin3a (sc-994, Santa Cruz Biotechnology), to Ing2 (ab109504, Abcam), to RbAp46/48 (39199, Active Motif), to AcH3K9 (39917, Active Motif), to BrdU (347580, BD Biosciences), and to 5hmC (39769, Active Motif).

Techniques: DNA Methylation Assay, Activity Assay, Immunofluorescence, Staining, Expressing, Fluorescence

Journal: eLife

Article Title: Loss of Fam60a, a Sin3a subunit, results in embryonic lethality and is associated with aberrant methylation at a subset of gene promoters

doi: 10.7554/eLife.36435

Figure Lengend Snippet:

Article Snippet: Additional antibodies included control rabbit IgG (Kamiya Biomedical or Thermo Fisher Scientific), conformation-specific mouse monoclonal antibodies to rabbit IgG (#3678, Cell Signaling) that can avoid detection of denatured rabbit IgG used for immunoprecipitation, as well as rabbit polyclonal antibodies to GFP (598, MBL International), to Hdac1 (ab31263, Abcam), to FLAG (F3165, Sigma-Aldrich), to Hdac2 (ab7029, Abcam), to Sin3a (sc-994, Santa Cruz Biotechnology), to Ing2 (ab109504, Abcam), to RbAp46/48 (39199, Active Motif), to AcH3K9 (39917, Active Motif), to BrdU (347580, BD Biosciences), and to 5hmC (39769, Active Motif).

Techniques: Recombinant, Methylation, Conjugation Assay, Software

Journal: Molecular Cell

Article Title: The Nucleosome Remodeling and Deacetylation Complex Modulates Chromatin Structure at Sites of Active Transcription to Fine-Tune Gene Expression

doi: 10.1016/j.molcel.2018.06.003

Figure Lengend Snippet: Mbd3 Induction Restores NuRD Activity to Mbd3 -Null ESCs (A) Model of the induction system: Mbd3 -null ESCs (left) contain ER-Mbd3b-ER (green diamonds, ER-M3b-ER) in the cytoplasm. Upon addition of tamoxifen, ER-M3b-ER enters the nucleus. (B) Nuclear extracts from Mbd3-inducible ESCs at different times after tamoxifen addition or from wild-type ESCs (WT) were probed with antibodies indicated at right. In the α-Mbd3 panel, the location of the ER-Mbd3b-ER transgene is indicated with an arrow, as are the locations of endogenous Mbd3 isoforms in WT cells. Sin3A and Pcna act as loading controls. Protein sizes are shown at left in kilodaltons. (C) Chd4 was immunoprecipitated from nuclear protein extracts across tamoxifen induction time course and probed with antibodies indicated at right. IgG, immunoglobulin G (IgG) control; Input, 10% input; IP, Chd4 immunoprecipitation. Protein sizes are shown at left in kilodaltons. (D) ChIP-qPCR for Chd4 (blue lines), Mbd3 (black lines), and IgG (gray lines) across the promoter and an enhancer of NuRD target gene Ppp2r2c at 0 and 24 hr of tamoxifen treatment. x axes show locations relative to the annotated transcription start site of Ppp2r2c . N ≥ 3 biological replicates. See also Figure S2 C. (E) ChIP-qPCR at the peak of Mbd3 ChIP signal in (D) plotted across a time course of tamoxifen addition. Significant enrichment of Mbd3 relative to no tamoxifen occurs from 15 min onward ( ∗∗∗∗ p < 0.0001, ∗∗ p < 0.01, and ∗ p < 0.05 using a two-tailed t test). N ≥ 3 biological replicates. (F) Input-normalized ChIP signal across WT Mbd3 peaks plotted for ER-ChIP in uninduced cells (left) or after 48 hr of tamoxifen treatment (middle) and for Mbd3-ChIP in WT ESCs (right). Mean signal is plotted across the top. (G) qRT-PCR using nascent RNA of indicated genes over 48 hr of the time course of tamoxifen addition (mean of relative expression ± SEM; plotted relative to time 0). Tamoxifen was added to either the Mbd3 inducible line (in red) or to the parental, Mbd3 -null line as a control (in black). N ≥ 3 biological replicates. (H) Unsupervised clustering of nascent RNA-seq during the Mbd3 induction time course. Significant changes (|FC| > 2; p < 0.05) in transcript level compared to time 0 are shown by gray bars in the left-hand panels. Triplicate samples were prepared and sequenced for each time point. See also , , and .

Article Snippet: Sin3a, rabbit polyclonal , Santa Cruz , RRID: AB_2187760.

Techniques: Activity Assay, Immunoprecipitation, Control, ChIP-qPCR, Two Tailed Test, Quantitative RT-PCR, Expressing, RNA Sequencing

Journal: Molecular Cell

Article Title: The Nucleosome Remodeling and Deacetylation Complex Modulates Chromatin Structure at Sites of Active Transcription to Fine-Tune Gene Expression

doi: 10.1016/j.molcel.2018.06.003

Figure Lengend Snippet:

Article Snippet: Sin3a, rabbit polyclonal , Santa Cruz , RRID: AB_2187760.

Techniques: Recombinant, RNA Sequencing

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: Sedimentation and Immunoprecipitation Assays for Analyzing Complexes that Repress Transcription

doi: 10.1007/978-1-62703-284-1_30

Figure Lengend Snippet: Detection of the putative co-repressor protein Gon4-like and potential interacting factors in sucrose gradient fractions containing high molecular weight species. Whole cell lysates were prepared from M12 B cells and separated through a 20–50% continuous sucrose gradient via ultracentrifugation as described in Methods. After centrifugation, the fractions indicated across the top of the panel (odd numbers only are shown) were collected and analyzed by immunoblot using the antibodies listed on the right side of the panels. M12 whole cell lysate (WCL) was used as a positive control for immunoblots (left side of panels). Protein standards were separated in parallel gradients and fractions containing these are indicated at the top of the panel. The box with the asterisk on top denotes fractions that contain Gon4-like, the co-repressor Sin3a, HDAC1 and the DNA-binding protein YY1, suggesting these factors associate as components of high-molecular weight complexes. Data shown are representative of 3 independent experiments.

Article Snippet: Affinity-purified rabbit polyclonal antibodies specific for Sin3a or non-specific rabbit IgG (both obtained from Santa Cruz Biotechnology) were used to perform immunoprecipitations.

Techniques: High Molecular Weight, Centrifugation, Western Blot, Positive Control, Binding Assay