Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A) Schematic drawing of CHD3, CHD4 and CHD5 proteins showing conserved domains and the CHD5 specific sequence. (B) Table showing sequence identity between different domains of CHD3, CHD4 and CHD5. (C) Expression of recombinant CHD5. Recombinant CHD5-Flag was purified from extracts of baculovirus-infected Sf9 cells. Extracts were incubated with anti-Flag affinity resin, washed and eluted with an excess of Flag peptide. Purified protein was seperated by SDS-PAGE followed by Western blotting with CHD5 antibody (lane 1) or silver staining (lane 2). Asterisk indicates degradation products with intact anti-CHD5 epitope. (D) CHD5 is a nucleosome-stimulated ATPase. Purified recombinant CHD5-Flag was subjected to ATPase assay as described in Materials and Methods. Different amounts of recombinant human CHD5-Flag, naked DNA or in vitro assembled nucleosomes were used as indicated at the bottom of the panel. Reaction products were separated by thin layer chromatography and the amount of released 32 P i was quantitated. The percentage of ATP hydrolysis was calculated for each reaction. The percentage of ATP hydrolysis of control reactions (without enzyme) was set to one. Error bars depict the standard deviation of 3 independent experiments.

Article Snippet: Western blotting of extracts was performed according to standard procedures using the primary antibodies anti mouse-CHD5 rabbit polyclonal (Santa Cruz Biotechnology (M-182) sc-68389) and anti Lamin B goat polyclonal (Santa Cruz Biotechnology (M-20) sc-6217).

Techniques: Sequencing, Expressing, Recombinant, Purification, Infection, Incubation, SDS Page, Western Blot, Silver Staining, ATPase Assay, In Vitro, Thin Layer Chromatography, Control, Standard Deviation

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A) CHD5 protein is expressed in mouse brain and testis. Western blot was performed with CHD5 antibody on lysates from different mouse tissues. Extracts from HEK cells transiently transfected with CHD5-Flag served as a positive control. Lamin B was used as a loading control. (B) Two different CHD5 isoforms are expressed in mouse brain and testis. Panel 1: schematic representation of the location of the PCR primers used to detect CHD5 cDNA of isoforms 1 and 2. Panel 2: schematic representation of part of the CHD5 gene structure showing the two different putative splicing variants. Exons 23, 24 and 25 are indicated. Horizontal lines are introns. Diagonal lines indicate splicing. Panel 3: RT-PCR detecting expression of different CHD5 isoforms in mouse testis and brain. H 2 O: negative control. Asterisk: primer dimers.

Article Snippet: Western blotting of extracts was performed according to standard procedures using the primary antibodies anti mouse-CHD5 rabbit polyclonal (Santa Cruz Biotechnology (M-182) sc-68389) and anti Lamin B goat polyclonal (Santa Cruz Biotechnology (M-20) sc-6217).

Techniques: Western Blot, Transfection, Positive Control, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Negative Control

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: Mouse brain sections were immunostained with antibodies against CHD5 or CHD3/4 and antibody binding was detected using a peroxidase labeled secondary antibody and 3,3'-diaminobenzidine (DAB) as a substrate. (1) CHD5 positive neurons in the hippocampus (20× magnification). (2) 200× magnification of CHD5 positive hippocampal neurons. (3) CHD5 positive neurons in the cortex of the cerebellum (100× magnification). (4) 400× magnification of CHD5 positive cerebellar neurons. (5) and (6) consecutive sections of cerebellum showing CHD3/4 (5) and CHD5 (6) expression.

Article Snippet: Western blotting of extracts was performed according to standard procedures using the primary antibodies anti mouse-CHD5 rabbit polyclonal (Santa Cruz Biotechnology (M-182) sc-68389) and anti Lamin B goat polyclonal (Santa Cruz Biotechnology (M-20) sc-6217).

Techniques: Binding Assay, Labeling, Expressing

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A) Western blot analysis CHD3/4, CHD5 and lamin expression in lysates from 3 week-old and from adult mice. Extracts from HEK cells expressing recombinant CHD5-Flag and extracts from murine brain were used as positive controls for CHD5 expression. Antibodies used for Western blot are indicated. (B) CHD5 protein is maximally expressed in round spermatids at Stages VII–VIII of the spermatognenic cycle. Upper images show results of the immunostaining and the bottom images show the PAS staining pattern of a section directly adjacent to the section shown above it. Left panel: low magnification (scale bars indicate 0.5 mm) of a mouse testis containing seminiferous tubules in different spermatogenic stages. Middle and right: higher magnification of the areas (scale bars indicate 0.05 mm) marked in the left panel, roman numerals indicate the stages of the spermatogenic cycle. (C) CHD5 is expressed in postmeiotic cells. Immunofluorescence analysis was performed using CHD5 antibody and visualized with confocal microscopy. Panel 1: Nuclei stained with DAPI. Panel 2: CHD5 staining, the outline of the tubule as seen in panel 1 is indicated with white lines. Panel 3: phosphorylated H2AX (γH2AX) staining. Spermatocytes in late meiotic prophase (pachytene and diplotene), show intense staining in a subregion of the nucleus (XY body) (arrow). Earlier spermatocytes (at the more basal part of the tubule) show nucleus wide γH2AX staining (arrowhead). Panel 4: overlay of the DAPI, CHD5 and γH2AX channels. (D) CHD5 and CHD3/4 expression at different stages of spermatogenesis. Panel 1: CHD5 is expressed in post-meiotic cells (100× magnification). Panel 2: CHD3/4 is expressed in the area where spermatogonia, Sertoli cells and spermatocytes are present (100× magnification). Panel 3: CHD5 immunostaining (250× magnification). Panel 4: CHD3/4 immunostaining (250x). Formalin-fixed paraffin-embedded tissue sections were immunostained with the antibodies indicated.

Article Snippet: Western blotting of extracts was performed according to standard procedures using the primary antibodies anti mouse-CHD5 rabbit polyclonal (Santa Cruz Biotechnology (M-182) sc-68389) and anti Lamin B goat polyclonal (Santa Cruz Biotechnology (M-20) sc-6217).

Techniques: Western Blot, Expressing, Recombinant, Immunostaining, Staining, Immunofluorescence, Confocal Microscopy, Formalin-fixed Paraffin-Embedded

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A, B) Spread nuclei of spermatids immunostained for CHD5 and counterstained with DAPI. An example of a round spermatid nucleus that is positive for CHD5 expression and displays the high intensity focus in the chromocenter is shown in A and such an example is also indicated with a white arrow in B, the yellow arrow indicates a spermatid nucleus that expresses CHD5, but does not have the extra CHD5 focus. The green arrow indicates a CHD5-negative round spermatid nucleus. (C) Spread nuclei of spermatids immunostained for CHD5 and H3K9me2, and counterstained with DAPI. The nuclear area encompassing the chromatin of the sex chromosome is indicated with a dashed line H3K9me2 marks the chromocenter and the sex chromosome in the left (early) round spermatid nucleus, and mainly the sex chromosome in the right (late) round spermatid nucleus. CHD5 level is very low in the left nucleus, but clearly enriched in the chromocenter and the focus (indicated with a white arrow) adjacent to the chromocenter and Y chromosome chromatin in the right nucleus.

Article Snippet: Western blotting of extracts was performed according to standard procedures using the primary antibodies anti mouse-CHD5 rabbit polyclonal (Santa Cruz Biotechnology (M-182) sc-68389) and anti Lamin B goat polyclonal (Santa Cruz Biotechnology (M-20) sc-6217).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Differential Expression and Sex Chromosome Association of CHD3/4 and CHD5 during Spermatogenesis

doi: 10.1371/journal.pone.0098203

Figure Lengend Snippet: (A,B) Spread wild type (A) and Spo11 null ( Spo11 YF/YF ) (B) spermatocyte nuclei stained for MacroH2A.1 and CHD3/4. Colocalisation on the PAR and the X centromere is observed in pachytene, but not in diplotene nuclei of wild type spermatocytes. In the absence of functional SPO11, some zygotene-like nuclei show enrichment of both CHD4 and macroH2A.1 in the pseudo XY body. (C) Spread spermatid nucleus stained for CHD5, and MacroH2A, colocalisation of the CHD5/MacroH2A focus in the chromocenter is clear in the merge with DAPI (right image). (D) Spread spermatid nuclei stained with macroH2A (green) and CREST (red, left panel) or with CHD5 and CREST (right panel). The enlargements in the middle of each panel clearly show that the macroH2A.1/CHD5 spot in the chromocenter does not colocalise with CREST marking of the centromeres. (E) Spread spermatid nuclei costained for CHD5 and RNA polymerase II. Both high and low levels of RNA polymerase II can be observed in nuclei that express CHD5.

Article Snippet: Western blotting of extracts was performed according to standard procedures using the primary antibodies anti mouse-CHD5 rabbit polyclonal (Santa Cruz Biotechnology (M-182) sc-68389) and anti Lamin B goat polyclonal (Santa Cruz Biotechnology (M-20) sc-6217).

Techniques: Staining, Functional Assay

Journal: iScience

Article Title: Chromodomain helicase DNA-binding domain 2 maintains spermatogonial self-renewal by promoting chromatin accessibility and mRNA stability

doi: 10.1016/j.isci.2022.105552

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-CHD2 , ABclonal , Cat# A5895 Lot: 0203250201 RRID: AB_2766643.

Techniques: Recombinant, Embryo Culture, CCK-8 Assay, Staining, TUNEL Assay, Apoptosis Assay, Mass Spectrometry, Plasmid Preparation, Software

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Log2 fold change (log2fc) of spatial distance between adjacent TADs versus -log10 false discovery rate (FDR) for each perturbation. Each dot represents a perturbation in the screen library. In all volcano plots, the top hits (nuclear proteins with the largest log2fc and FDRs<0.1) in both directions are indicated with blue (knockout leads to upregulation) and red dots (knockout leads to downregulation), respectively. The top candidate genes which when knocked out led to increased adjacent TAD distances are: RB1, MRVI1 and PIP5K1B; the top candidate genes which when knocked out caused decreased adjacent TAD distances are: GLDC, NR4A1 and ZNF114. Positive controls (NIPBL and CTCF) are marked in black. ( B ) Log2 fold change of adjacent TAD distance across chr22 for selected hits. ( C ) Spatial distances between adjacent TADs for non-targeting control and selected hits. ( D ) Log2 fold change of long-range A-A contact frequency versus -log10 FDR for each perturbation. Top three hits in both directions including NR4A1, PDE1A, HOXB9, RB1, PCBP1 and LRRC10B are labeled. ( E ) Long-range A-A contact frequencies for non-targeting control and selected hits. ( F ) Log2 fold change of long-range A-B contact frequency versus -log10 FDR for each perturbation. Top three hits in both directions, including RFESD, HOXB9, FAM69B, C2CD2, CHD7 and FAM13C, are labeled. ( G ) Long-range A-B contact frequencies for non-targeting control and selected hits. ( H ) Log2 fold change of long-range B-B contact frequency versus -log10 FDR for each perturbation. Top hits in both directions, including FOS, NR4A1, DDX24 and MYBPH, are labeled. ( I ) Long-range B-B contact frequencies for non-targeting control and selected hits. ( J ) Log2 fold change of overall inter-TAD distances versus -log10 FDR for each perturbation. Top three hits in both directions, including PCBP1, RB1, CHD7, GLDC, HOXB9 and CUL1, are labeled. ( K ) Overall inter-TAD distances for non-targeting control and selected hits. ( L ) Log2 fold change of individual overall inter-TAD distances in chr22 for selected hits. P values in (C) and (K) were calculated by two-sided Wilcoxon signed rank test. P values in (E), (G) and (I) were calculated by two-sided Wilcoxon rank sum test. In all box plots throughout the manuscript, the boxes cover the 25 th to 75 th percentiles, the whiskers cover the 10 th to 90 th percentiles, and the line in the middle of the boxes represents the median value. For all relevant panels, significance is represented as *p<0.1. **p<0.05. ***p<0.01.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Knock-Out, Labeling

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Western blot of siCtrl- and siCHD7-treated A549-Cas9 nuclear extracts. Top: anti-CHD7 antibody; bottom: anti-Actin B antibody. ( B ) A-B compartment profile of chr22 in siCtrl cells. ( C ) A-B compartment profile of chr22 in siCHD7 cells. ( D ) Polarization indices of chr22 A-B compartments of siCtrl (white) and siCHD7 (orange). Shadowed boxes show the polarization indices from randomized controls, where the compartment identities of TADs are scrambled. ( E ) Long-range contact frequency of siCtrl and siCHD7 (shadowed) among A compartments (red), between A and B compartments (purple), and among B compartments (blue). ( F ) Overall inter-TAD distance of siCtrl and siCHD7. ( G ) Radii of gyration of siCtrl and siCHD7. P values in (D), (E) and (G) were calculated by two-sided Wilcoxon rank sum test. P value in (F) was calculated by two-sided Wilcoxon signed rank test.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Western Blot

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Log2 fold change of inter-TAD distance of siCHD7 compared to siCtrl. Number of traces analyzed: 3,558 (siCtrl), 4,134 (siCHD7). ( B ) Log2 fold change of short-range (defined as spatial distances between genomic regions that are less than 3Mb apart) and long-range (defined as spatial distances between genomic regions that are more than 3Mb apart) inter-TAD distances between siCHD7 and siCtrl. ( C ) Log2 fold change of inter-TAD distance of CHD7 overexpression compared to GFP overexpression. Number of traces analyzed: 3,157 (GFP OE), 1,174 (CHD7 OE). ( D ) Log2 fold change of short-range and long-range inter-TAD distances between CHD7 and GFP overexpression. ( E ) Log2 fold change of inter-TAD distance of TSA-treated cells compared to DMSO-treated cells. Number of traces analyzed: 1,214 (DMSO), 2,223 (TSA). ( F ) Log2 fold change of short-range and long-range inter-TAD distances between cells with TSA and DMSO treatment. ( G ) Log2 fold change of inter-TAD distance of siCTCF compared to siCtrl. Number of traces analyzed: 5,768 (siCtrl), 4,226 (siCTCF). ( H ) Log2 fold change of short-range and long-range inter-TAD distances between siCTCF and siCtrl. ( I ) Log2 fold change of inter-TAD distance of double knockdown of CTCF and CHD7 (doubleKD) compared to siCTCF. Number of traces analyzed: 3,560 (doubleKD). ( J ) Log2 fold change of short-range and long-range inter-TAD distances between doubleKD and siCTCF. ( K ) Working model of CHD7 compacting chromatin over long range (cartoon created with Biorender.). All chromatin tracing experiments in this figure were done in the A549 cell background, targeting chr22. P values were calculated by two-sided Wilcoxon signed rank test. For all violin plots, the median log2 fold change values are indicated by colored dashed line, the quartiles are indicated by colored dotted line, and value 0 is marked by black dashed line.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Over Expression

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) A-B compartment profile of chr22 in A549-Cas9 cells with GFP overexpression. ( B ) A-B compartment profile of chr22 in A549-Cas9 cells with CHD7 overexpression. ( C ) Polarization indices of cells with GFP (white) and CHD7 (orange) overexpression and the according randomized controls (shadowed). ( D ) Long-range contact frequency of cells with GFP of CHD7 (shadowed) overexpression in A compartments (red), across A and B compartments (purple) and in B compartments (blue). ( E ) Overall inter-TAD distance of chr22 in cells with GFP and CHD7 overexpression. ( F ) Radii of gyration of chr22 in cells with GFP and CHD7 overexpression. P values in (C), (D) and (F) were calculated by two-sided Wilcoxon rank sum test. P value in (E) were calculated by two-sided Wilcoxon signed rank test.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Over Expression

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Top 10 gene ontology terms up and down in siCHD7 cells versus siControl cells. Gene ontology was performed using Enrichr. ( B ) Volcano plot of RNA-seq of siCHD7 and siControl. Top differentially expressed genes are displayed on the graph as labels. CHD7 is highlighted and is a top differentially downregulated gene in the siCHD7 cells, validating the knockdown. ( C ) Heat map of other proteins/epigenetic mark localized to CHD7 peaks by CUT&RUN. ( D ) Example tracks of CUT&RUN peak profiles of CHD7 and other proteins/epigenetic mark over a gene. ( E ) Peak annotation for all CHD7 peaks. ( F ) Overlap of CUT&RUN peaks of CTCF, RAD21, and H3K4me3 with CHD7 peaks on promoter regions.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: RNA Sequencing Assay

Journal: bioRxiv

Article Title: High-content image-based CRISPR screening reveals regulators of 3D genome architectures

doi: 10.1101/2023.01.31.525983

Figure Lengend Snippet: ( A ) Western blot of siCtrl, siCHD7, siCTCF and doubleKD (siCHD7+siCTCF) in A549-Cas9 cells. Top: anti-CHD7 antibody; middle: anti-CTCF antibody; bottom: anti-Actin B antibody. ( B ) Short-range inter-TAD distance of chr22 in siCtrl, siCTCF and doubleKD cells. ( C ) Long-range inter-TAD distance of chr22 in siCtrl, siCTCF and doubleKD cells. ( D ) Overall inter-TAD distance of chr22 in siCtrl, siCTCF and doubleKD cells. ( E ) Radii of gyration of chr22 in siCtrl, siCTCF and doubleKD cells. P values in (B) to (D) were calculated by two-sided Wilcoxon signed rank test. P values in (E) was calculated by two-sided Wilcoxon rank sum test.

Article Snippet: H2A antibody – CST Histone H2A (D6O3A) Rabbit mAb #12349 IgG Control antibody – CUTANA Kit Rabbit IgG CUT&RUN Negative Control Antibody H3K4me3 antibody – CST Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 CTCF antibody – CTCF CUTANA™ CUT&RUN Antibody 13-2014 CHD7 antibody – Invitrogen CHD7 Polyclonal Antibody PA5-72964 RAD21 antibody – Active Motif Rad21 #91245

Techniques: Western Blot

Journal: Cell reports

Article Title: Chd8 mutation leads to autistic-like behaviors and impaired striatal circuits

doi: 10.1016/j.celrep.2017.03.052

Figure Lengend Snippet: (A) Bar chart of CHD8 binding peaks as a percentage of total peaks showing CHD8 primarily binds in promoters (WT 38 %; Chd8+/− 39 %) for both genotypes. Replicate somatosensory cortices from wild-type (n = 2) and Chd8+/− mice (n = 2) were microdissected and used for ChIP-seq. ChIP-Seq controls were both input and IgG for each genotype. Peaks were called for each genotype and each control using MACS2 (FDR cutoff of 1%). Only peaks shared between input and IgG for a particular genotype were considered. Annotations were made using HOMER with the mouse mm10 genome assembly and annotation.

Article Snippet: Chromatin fragments were then incubated overnight at 4°C with anti-CHD8 antibody (Novus Biologicals, NB100-60417).

Techniques: Binding Assay, ChIP-sequencing, Control

Journal: Cell reports

Article Title: Chd8 mutation leads to autistic-like behaviors and impaired striatal circuits

doi: 10.1016/j.celrep.2017.03.052

Figure Lengend Snippet: (A) Table of the top 10 upregulated (top) and downregulated (bottom) differentially expressed genes. Differential expression analysis using DEseq2 was performed on a TPM expression matrix from RNA sequencing libraries generated from different brain regions when comparing 10-week old male Chd8+/− mice and wild-type littermates. Also see Figure S4.

Article Snippet: Chromatin fragments were then incubated overnight at 4°C with anti-CHD8 antibody (Novus Biologicals, NB100-60417).

Techniques: Quantitative Proteomics, Expressing, RNA Sequencing, Generated

Journal: Cell reports

Article Title: Chd8 mutation leads to autistic-like behaviors and impaired striatal circuits

doi: 10.1016/j.celrep.2017.03.052

Figure Lengend Snippet: (A) (Top) Representative sEPSC traces from MSNs in the core region of the NAc of Chd8+/− mice and wild-type littermates. Chd8+/− mice displayed both an increase in (Left) sEPSC frequency [WT (n = 24) 5.0 ± 0.4 Hz SEM; Chd8+/− (n = 28) 6.3 ± 0.5 Hz SEM, two-tailed t-test p-value = 0.048] as well as (Right) sEPSC amplitude [WT (n = 24) 16.3 ± 0.5 pA SEM; Chd8+/− (n = 28) 19.2 ± 0.8 pA SEM, two-tailed t-test p-value = 0.006] compared to wild-type littermates.

Article Snippet: Chromatin fragments were then incubated overnight at 4°C with anti-CHD8 antibody (Novus Biologicals, NB100-60417).

Techniques: Two Tailed Test

Journal: Cell reports

Article Title: Chd8 mutation leads to autistic-like behaviors and impaired striatal circuits

doi: 10.1016/j.celrep.2017.03.052

Figure Lengend Snippet: (A) During juvenile social play we observed no difference in the total number of all interactive events between Chd8+/− and wild-type littermate mouse pairs [WT (n = 15) 107 ± 6 events SEM; Chd8+/− (n = 17) 114 ± 8 events SEM, one-way ANOVA with Bonferroni post hoc test p-value > 0.05]. Also see Figure S5.

Article Snippet: Chromatin fragments were then incubated overnight at 4°C with anti-CHD8 antibody (Novus Biologicals, NB100-60417).

Techniques:

Journal: Cell reports

Article Title: Chd8 mutation leads to autistic-like behaviors and impaired striatal circuits

doi: 10.1016/j.celrep.2017.03.052

Figure Lengend Snippet: (A) (Left) Open field traces for animals with median center time values. (Right) In the open field test, Chd8+/− mice spent less time in the center compared to wild-type littermates [WT (n = 55) 360 ± 30 s SEM; Chd8+/− (n = 64) 190 ± 20 s SEM, two-tailed t-test p-value < 0.001]. Also see Figure S6.

Article Snippet: Chromatin fragments were then incubated overnight at 4°C with anti-CHD8 antibody (Novus Biologicals, NB100-60417).

Techniques: Two Tailed Test

Journal: The EMBO Journal

Article Title: Phosphoproteomic analysis reveals that PP4 dephosphorylates KAP-1 impacting the DNA damage response

doi: 10.1038/emboj.2012.86

Figure Lengend Snippet: PP4C influences the phosphorylation status of multiple DDR proteins. (A) Schematic for phosphoproteomics-based identification of putative PP4C substrates. (B) Identification of regulated phosphorylation in response to PP4C depletion. The geometric mean of iTRAQ spectral peak height ratios for replicate samples was plotted as a function of the sum of the geometric means of all iTRAQ spectral peak heights. Maximum approximate conditional likelihood (MACL) was used to determine an intensity-based variance function from which the 95% acceptance region was calculated (grey curve and Supplementary Table 1). Several DDR proteins, including KAP1, CHD4 and TP53BP1, exhibited hyperphosphorylation in response to deplection of PP4C. (C) Validation of the PP4 targets. (Left panel) HeLa S3 cells transfected with PP4C or scrambled siRNAs were exposed to IR, lysed after 2 h and immunoprecipitated (IP) using a pan-phosphoSer antibody and probed for DDR proteins as indicated. The relative band intensities are provided below each immunoblot. (Right panel) Proteins identified as putative PP4 substrates were confirmed using Phos–tag. HeLa S3 cells were transfected with PP4C siRNA and irradiated with 10-Gy IR. Lysates were subjected to SDS–PAGE containing 20 μM Phos–tag and immunobloted with indicated antibodies. Lysates treated with λ protein phosphatase (λPP) served as control for the Phos–tag-induced mobility shift.

Article Snippet: Antibodies used were against KAP-1 (BD Transduction Laboratories), pS824-KAP-1 (Bethyl), pS473-KAP-1 (BioLegend), 53BP1 (Cell signaling), CHD3 (Bethyl), CHD4 (Bethyl), RPA2 (Cell Signaling), PP4R1 (Bethyl), PP4R2 (Bethyl), PP4R3α (Bethyl), PP4R3β (Bethyl), PP4C (Bethyl), PP1α (Bethyl), PP1β (Bethyl), CHK1 (Cell Signaling), CHK2 (Cell Signaling), Flag-tag (Sigma), α-tubulin (Sigma) and Phoshphoserine-agarose conjugate (Abcam).

Techniques: Transfection, Immunoprecipitation, Western Blot, Irradiation, SDS Page, Mobility Shift

Journal: Cell reports

Article Title: Impact of WIN site inhibitor on the WDR5 interactome

doi: 10.1016/j.celrep.2020.108636

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-CHD8 , Bethyl Laboratories , Cat# A301–224A; RRID:AB_890578.

Techniques: Western Blot, Virus, Recombinant, Protease Inhibitor, Multiplex sample analysis, Staining, Cloning, Expressing, Transfection, Mass Spectrometry, Plasmid Preparation, Mutagenesis, CRISPR, Amplification, Modification, Software, Hybridization, Membrane

Journal: eLife

Article Title: Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

doi: 10.7554/eLife.37663

Figure Lengend Snippet: ( A,C ) HEK293T cells were treated with siRNAs targeting CHD3 ( A ), MSI1 ( B ), or TRIM32 ( C ), or non-targeting scramble siRNAs, then transfected with either empty vector or muSOX and subjected to chromatin immunoprecipitation (ChIP) using antibodies to RNAPII or IgG. Purified chromatin was quantified by qPCR. Western blots showing the levels of CHD3 and MSI1 after siRNA depletion, along with a GAPDH or histone H3 loading control are shown below. Graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005.

Article Snippet: Nuclear, cytoplasmic, and whole cell lysates were quantified by Bradford assay and resolved by SDS-PAGE and western blotted with antibodies against PABPC (Cell Signaling, 1:1000), PABPC4 (Bethyl, 1:1000), LARP4 (Thermo Fisher, 1:1000), Gapdh (Abcam, 1:3000), Histone H3 (Cell Signaling, 1:2000), LYRIC (Abcam, 1:1000), RRBP1 (Bethyl, 1:1000), MSI1 (Abcam, 1:1000), Lin28b (Abcam, 1:1000), CHD3 (Cell Signaling, 1:1000), RPP20 (Novus, 1:1000), THOC6 (Life Technologies, 1:1000), PNN (Life Technologies, 1:1000), EXO4 (rabbit polyclonal produced using recombinant EXO4 with an MBP tag, 1:1000), NPM (Abcam, 1:1000), GW182 (Abcam, 1:1000), DDX6 (Bethyl, 1:1000), DCP2 (Bethyl, 1:1000), TRIM32 (Abcam, 1:1000), RNAPII Rpb1 (BioLegend, 1:2000), TBP (Abcam, 1:2000).

Techniques: Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Purification, Western Blot

Journal: eLife

Article Title: Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

doi: 10.7554/eLife.37663

Figure Lengend Snippet: ( A–E ) HEK293T cells were treated with siRNAs targeting PABPC1 and PABPC4 ( A ), LARP4 ( B ), CHD3 ( C ), MSI1 ( D ), or TRIM32 ( E ), and subjected to ChIP using antibodies to RNAPII or IgG. Purified chromatin was quantified by qPCR. All graphs display individual biological replicates as dots, with the mean and SEM. Statistical significance was determined using Student’s t test *p<0.05 **p<0.005 ***p<0.0005.

Article Snippet: Nuclear, cytoplasmic, and whole cell lysates were quantified by Bradford assay and resolved by SDS-PAGE and western blotted with antibodies against PABPC (Cell Signaling, 1:1000), PABPC4 (Bethyl, 1:1000), LARP4 (Thermo Fisher, 1:1000), Gapdh (Abcam, 1:3000), Histone H3 (Cell Signaling, 1:2000), LYRIC (Abcam, 1:1000), RRBP1 (Bethyl, 1:1000), MSI1 (Abcam, 1:1000), Lin28b (Abcam, 1:1000), CHD3 (Cell Signaling, 1:1000), RPP20 (Novus, 1:1000), THOC6 (Life Technologies, 1:1000), PNN (Life Technologies, 1:1000), EXO4 (rabbit polyclonal produced using recombinant EXO4 with an MBP tag, 1:1000), NPM (Abcam, 1:1000), GW182 (Abcam, 1:1000), DDX6 (Bethyl, 1:1000), DCP2 (Bethyl, 1:1000), TRIM32 (Abcam, 1:1000), RNAPII Rpb1 (BioLegend, 1:2000), TBP (Abcam, 1:2000).

Techniques: Purification

Journal: eLife

Article Title: Changes in mRNA abundance drive shuttling of RNA binding proteins, linking cytoplasmic RNA degradation to transcription

doi: 10.7554/eLife.37663

Figure Lengend Snippet:

Article Snippet: Nuclear, cytoplasmic, and whole cell lysates were quantified by Bradford assay and resolved by SDS-PAGE and western blotted with antibodies against PABPC (Cell Signaling, 1:1000), PABPC4 (Bethyl, 1:1000), LARP4 (Thermo Fisher, 1:1000), Gapdh (Abcam, 1:3000), Histone H3 (Cell Signaling, 1:2000), LYRIC (Abcam, 1:1000), RRBP1 (Bethyl, 1:1000), MSI1 (Abcam, 1:1000), Lin28b (Abcam, 1:1000), CHD3 (Cell Signaling, 1:1000), RPP20 (Novus, 1:1000), THOC6 (Life Technologies, 1:1000), PNN (Life Technologies, 1:1000), EXO4 (rabbit polyclonal produced using recombinant EXO4 with an MBP tag, 1:1000), NPM (Abcam, 1:1000), GW182 (Abcam, 1:1000), DDX6 (Bethyl, 1:1000), DCP2 (Bethyl, 1:1000), TRIM32 (Abcam, 1:1000), RNAPII Rpb1 (BioLegend, 1:2000), TBP (Abcam, 1:2000).

Techniques: Transfection, Construct, Synthesized, Knock-Out, Stable Transfection, Expressing, Recombinant, Plasmid Preparation, Western Blot, Sequencing, Software