Journal: International Journal of Molecular Sciences

Article Title: Prion-Associated Neurodegeneration Causes Both Endoplasmic Reticulum Stress and Proteasome Impairment in a Murine Model of Spontaneous Disease

doi: 10.3390/ijms22010465

Figure Lengend Snippet: Ub G76V -GFP intracellular accumulation is observed in brain areas showing prion-associated neuropathology. ( A ) Hippocampus from a clinical TgU1 + /TgVole + mouse stained with hematoxylin and eosin and immunostained for Ub G76V -GFP and glial fibrillary acidic protein (GFAP). This animal shows severe spongiosis in the hippocampus and intense immunostaining for Ub G76V -GFP and GFAP. Strong Ub G76V -GFP immunolabeling is observed, affecting numerous cells that appear to be reactive astrocytes. ( B) Dual immunofluorescence staining with anti-GFAP and anti-GFP antibodies revealed that numerous reactive astrocytes accumulated the Ub G76V -GFP reporter, suggesting that these cells can compensate for proteasome impairment and accumulate high amounts of ubiquitin conjugates before succumbing to the cytotoxic effect.

Article Snippet: Immunodetection was performed overnight at 4 °C using a rabbit polyclonal anti-GFP primary antibody (1:2500; anti-GFP antibody-ChIP Grade, ab290, Abcam, Cambridge, United Kingdom).

Techniques: Staining, Immunostaining, Immunolabeling, Immunofluorescence, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from transcription sites

doi: 10.1101/395863

Figure Lengend Snippet: ( A ) Fluorescence imaging of tetramethylrhodamine conjugated Wheat Germ Agglutinin (WGA, red) and UPF1-GFP (green) in salivary gland cells. Lower panels are magnified view of boxed area in upper panels. ( B ) Imaging of WGA (red) in wild type (upper panel) and FkhGAL4>UPF1-RNAi (lower panel) salivary gland cells. Yellow arrow indicates nuclear envelope. Cells were counter-stained with DAPI (blue).

Article Snippet: These antibodies were used for immunostaining: mouse anti-UPF1 (described in this paper 7B12, typically diluted 1:100), mouse IgM anti-Ser2 Pol II (H5, Covance AB_10143905, 1:500), mouse anti-hnRNPA1 (Hrb87F, P11, 1:50) , mouse anti-GFP (B-2, Santa Cruz, SC-9996, 1:200), Tetramethylrhodamine Conjugate Wheat Germ Agglutinin (Thermo Fisher, W7024, 10µg/mL).

Techniques: Fluorescence, Imaging, Staining

Journal: bioRxiv

Article Title: The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from transcription sites

doi: 10.1101/395863

Figure Lengend Snippet: ( A ) Fluorescence immunolocalization of UPF1 using the 7B12 monoclonal antibody (Cy3, red) on polytene chromosomes (blue) of wild type (I, II) and UPF1-RNAi (III, IV) salivary glands. ( B ) Western blotting probed with 7B12 mab for protein extracts of 3 rd instar larval salivary gland from FkhGAL4>UPF1-GFP (lane I), wild type (lane II) and FkhGAL4>UPF1-RNAi (lane III). Ponceau staining of the same blot showing equal protein loading. ( C ) Immunolocalization of UPF1-GFP (FITC, green, I, III) on polytene chromosomes of FkhGAL4>UPF1-GFP salivary glands, detected using anti-GFP antibody. Chromosomes were counter stained with DAPI (blue, II, III). Line profiles in IV show both signal intensities along the white line traced on the chromosome arm in III. Note that UPF1 signal peaks at chromatin-decondensed regions characterised by low DAPI signal.

Article Snippet: These antibodies were used for immunostaining: mouse anti-UPF1 (described in this paper 7B12, typically diluted 1:100), mouse IgM anti-Ser2 Pol II (H5, Covance AB_10143905, 1:500), mouse anti-hnRNPA1 (Hrb87F, P11, 1:50) , mouse anti-GFP (B-2, Santa Cruz, SC-9996, 1:200), Tetramethylrhodamine Conjugate Wheat Germ Agglutinin (Thermo Fisher, W7024, 10µg/mL).

Techniques: Fluorescence, Western Blot, Staining

Journal: bioRxiv

Article Title: The RNA helicase UPF1 associates with mRNAs co-transcriptionally and is required for the release of mRNAs from transcription sites

doi: 10.1101/395863

Figure Lengend Snippet: ( A ) UPF1 (red) and Ser2 Pol II (pink) ChIP-seq enrichment profiles at RpL23A (on left) and RpS12 (on right) gene loci. ( B ) Real-time PCR quantification of average ChIP signal of either endogenous UPF1 (red) or GFP (as negative control, grey) at the RpL23 and RpS12 genes in salivary glands expressing GFP. The locations of the primer pairs used are indicated by the black boxes (P) shown below the genes schematics in A. ( C ) Real-time PCR quantification of average UPF1 association in control (red) or UPF1-RNAi (blue) S2 cells. ( D ) Real-time PCR quantification of average UPF1 association at three distinct regions of RpL23 in S2 cells (same primers pairs as in ) with (blue) or without (red) RNase A treatment. ( E ) Ser2 Pol II immunoprecipitation of S2 cell nuclear extracts using anti-Ser2 Pol II antibody (ab5095) and detection (same blot) of Ser2 Pol II, UPF1, eIF4AIII and hnRNPA1, in control (lanes 2-3) or RNase treated samples (lanes 4-5). IP refers to immunoprecipitated fractions, Ub to unbound fractions.

Article Snippet: These antibodies were used for immunostaining: mouse anti-UPF1 (described in this paper 7B12, typically diluted 1:100), mouse IgM anti-Ser2 Pol II (H5, Covance AB_10143905, 1:500), mouse anti-hnRNPA1 (Hrb87F, P11, 1:50) , mouse anti-GFP (B-2, Santa Cruz, SC-9996, 1:200), Tetramethylrhodamine Conjugate Wheat Germ Agglutinin (Thermo Fisher, W7024, 10µg/mL).

Techniques: ChIP-sequencing, Real-time Polymerase Chain Reaction, Negative Control, Expressing, Immunoprecipitation

Journal: bioRxiv

Article Title: HNF1A is a novel BRD4 target and critical for BET-inhibitor response in pancreatic ductal adenocarcinoma

doi: 10.1101/2025.10.01.679805

Figure Lengend Snippet: BRD4 directly regulates HNF1A expression. A, Western blotting for HNF1A, BRD2, BRD3, BRD4, and Actin for AsPC-1, HPAF-II, UM5, and UM15 cells transfected with control (Ctl), BRD2, BRD3, BRD4, or combined BET protein siRNAs for 96 hours. B, quantitative RT-PCR analysis of HNF1A mRNA levels following 96 hours of knockdown of AsPC-1, HPAF-II, UM5, and UM15 cells with Ctl or BRD4 siRNA. Bar graphs represent the mean ± SEM, n=3. Statistical difference was determined by two-sided Student t-test with Welch’s correction; ns = non-significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. C, UCSC Genome Browser ChIP-seq tracks for the HNF1A locus. ChIP-seq experiments for Histone H3 acetyl-lysine 27 and BRD4 are shown for HNF1A-positive (HepG2, A594, 22Rv1) and HNF1A-negative (K562, Panc1, HCT116) cells. The HNF1A proximal promoter region is indicated. D, ChIP-PCR was performed on AsPC-1 cells treated with DMSO or 0.5 µM OTX-015 for 24 hours using normal IgG or BRD4 antibody. Enrichment of the HNF1A promoter was compared to the second intron of HNF1A as an intragenic control and the MYOD promoter as an inactive promoter control. Bar graphs represent the mean ± SEM, n=3. Statistical difference was determined by two-sided Student t-test with Welch’s correction; ns = non-significant, *p<0.05. E, schematic representation of a HNF1A reporter construct. A 1kbp region of the proximal promoter region, including the 5’ UTR was cloned upstream of a GFP reporter. F, Western blotting of HNF1A, GFP, BRD4, and Actin for AsPC-1 reporter cells treated with DMSO, 0.5 µM OTX-015 or 0.1 µM ABBV-744 for 24 hours or transfected with Ctl or BRD4 siRNA for 96 hours. G, correlation of HNF1A and BRD4 mRNA from Moffitt et al ., 2015 . H, correlation of HNF1A and BRD4 mRNA from PDAC tumors from TNMplot.com .

Article Snippet: HNF1A (D7Z2Q; RRID: AB_2728751), HNF4A (C11F12; AB_2295208), HNF4G (E4V2B), MYC (D3N8F; RRID: AB_2631168), BRD2 (D89B4; RRID: AB_10835146), BRD4 (E1Y1P), phospho-RB (S807/811) (D20B12; RRID: AB_111786578), phospho-Histone H3 S10 (D7N8E; RRID: AB_2799431), and GFP (D5.1; RRID: AB_1196615) were purchased from Cell Signaling.

Techniques: Expressing, Western Blot, Transfection, Control, Quantitative RT-PCR, Knockdown, ChIP-sequencing, Construct, Clone Assay

Journal: Nature Communications

Article Title: Light regulates stomatal development by modulating paracrine signaling from inner tissues

doi: 10.1038/s41467-021-23728-2

Figure Lengend Snippet: a , b Gene expression analyses of STOMAGEN in WT, hy5-215 , hy5-51 (a only), and HY5-OX by RT-qPCR. In ( a ), RNA was extracted from 3-day-old seedlings grown under the light. In ( b ), seedlings were grown in darkness for 4 days and were exposed to light for 0, 2, and 4 h before harvest. Values are mean + /− SEM, n = 3 biological replicates. One-way ( a ) or two-way ( b ) ANOVA with Tukey’s multiple comparisons test, P < 0.01. c Co-expression of HY5 and STOMAGEN in the mesophyll layer. Confocal analysis of 3-day-old abaxial cotyledons of a transgenic seedling harboring both HY5pro:HY5-YFP and STOMAGENpro:H2B-mScarlet-I . From left: YFP signals (yellow), mScarlet-I signals (magenta), autofluorescence (cyan) and merged image of all three channels. Scale bar, 50 μm. Three independent cotyledons were examined with similar results ( d ) Gene structure of STOMAGEN . Arrow indicates the translational start site. Vertical bars mark the position of a Z-box (upstream of TSS only). P1 to 3 represent region(s) tested by EMSA ( e ), DNA pull down ( f ) and ChIP-qPCR ( g ). e EMSA analysis showing the binding of HY5 to a promoter fragment of STOMAGEN (P2). Recombinant MBP (control) and MBP-HY5 were assayed for binding with the biotin-labeled P2 probe. An unlabeled probe (competitor) was used to determine binding specificity (lanes 4–6). f DNA pull-down analysis showing the binding of HY5 to P2 and its dependence on the Z-box. Biotin-labeled probes, including a P2 probe with a mutated Z-box (mP2), were used to pull-down recombinant MBP (control) and MBP-HY5. Results were analyzed by western blotting using an anti-MBP antibody. g ChIP-qPCR assays were performed on WT and HY5pro:HY5-YFP using an anti-GFP antibody. Seedlings were grown for 4 days in darkness before exposed to light for 4 h or kept in the dark. Promoter regions of STOMAGEN (see d) were tested. A genomic region downstream of STOMAGEN and IR1 (see “Methods”) were used as negative controls. Values are mean + /− SEM, n = 3 technical replicates. Assay was repeated with similar results. h GUS reporter assay of two independent lines of STOMAGENpro:GUS and mSTOMAGENpro:GUS , which carries a mutated Z-box in the P2 region, in WT and hy5-215 . Seedlings were grown for 3 days under the light. Scale bar, 2 mm. Light intensity used: 200 ( a ) or 100 µmol m −2 s −1 ( b , c , g , h ). The experiments in ( e ) and ( f ) were carried out two times with similar results.

Article Snippet: Total proteins were extracted from seedlings using 1× Laemmli sample buffer (Bio-Rad, 1610737) and immunoblotting was carried out with an HRP-conjugated anti-GFP antibody (Miltenyi Biotec, 130-091-83, 1:3000 dilution) or a Rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956, 1:1000 dilution) followed by anti-rabbit IgG-HRP (Cell Signaling Technology, 7074, 1:3000 dilution).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Transgenic Assay, ChIP-qPCR, Binding Assay, Recombinant, Control, Labeling, Western Blot, Reporter Assay

Journal: Genome Research

Article Title: Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery

doi: 10.1101/gr.215186.116

Figure Lengend Snippet: Identification of chromatin located at the nuclear periphery by RE-ChIP. ( A ) Localization of the NUP1:GFP protein in an Arabidopsis nucleus: (scale bar) 2 µm. ( B ) Procedures for RE-mediated ChIP with NUP1:GFP (green). Chromatin (purple lines) fragmentation and isolation are conducted with a combination of RE (restriction enzyme) digestion and mild sonication. ( C ) Normalized sequence coverage (50-kb window size) on Chromosome 5 from various ChIP experiments. The horizontal bars depict pericentromeric regions, within which centromeric regions are highlighted in red. ( D ) NUP1:GFP RE-mediated ChIP-seq signal (50-kb window size), represented as the log 2 value of the ratio between normalized anti-GFP and IgG coverage, over all five chromosomes. Horizontal bars indicate the centromeric/pericentromeric regions, as in C .

Article Snippet: Next, the sheared chromatin was mixed with an equal volume of IP buffer (50 mM Hepes, pH 7.5; 150 mM NaCl; 5 mM MgCl 2 ; 10 μM ZnSO 4 ; 1% Triton X-100; 0.05% SDS) and then equally divided and incubated with anti-GFP antibody (Abcam, ab290) or normal rabbit IgG (Santa Cruz, sc-2027), respectively.

Techniques: Isolation, Sonication, Sequencing, ChIP-sequencing

Journal: Genome Research

Article Title: Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery

doi: 10.1101/gr.215186.116

Figure Lengend Snippet: Correlation between chromatin anchored at the nuclear periphery and the Hi-C map. ( A ) Correlation between NUP1:GFP RE-ChIP-seq signal and Hi-C map. The Hi-C maps (normalized at 20-kb resolution) of the left and right Chromosome 1 arms are shown as Spearman correlation matrices, from which PCA was conducted; the eigenvalues of the first component are plotted below (red and blue bars) together with the NUP1:GFP signal (green lines, 20-kb window size), represented as the log 2 value of the ratio between normalized anti-GFP and IgG coverage. ( B ) Anti-correlation between the telomeres and NUP1:GFP RE-ChIP-seq signal. The left panel shows a Spearman correlation matrix of Chromosome 3 derived from a Hi-C map at 20-kb resolution. Arrows depict KEE regions. The right panels highlight the 6-Mb distal chromosome regions, in which their correlation with the chromosome terminus (the first 20 kb of Chromosome 3) in the Hi-C map are shown as black curves. Green curves show the NUP1:GFP signal, as in A . Due to physical linkage, chromosome termini are expected to have strong colocalization with telomeres in the nucleus. In a Hi-C experiment, chromosome termini can be used to infer the spatial interactions between telomeres and other genomic regions.

Article Snippet: Next, the sheared chromatin was mixed with an equal volume of IP buffer (50 mM Hepes, pH 7.5; 150 mM NaCl; 5 mM MgCl 2 ; 10 μM ZnSO 4 ; 1% Triton X-100; 0.05% SDS) and then equally divided and incubated with anti-GFP antibody (Abcam, ab290) or normal rabbit IgG (Santa Cruz, sc-2027), respectively.

Techniques: Hi-C, ChIP-sequencing, Derivative Assay

Journal: Genome Research

Article Title: Nonrandom domain organization of the Arabidopsis genome at the nuclear periphery

doi: 10.1101/gr.215186.116

Figure Lengend Snippet: Genome-wide identification of NUP1-enriched regions in various tissues. ( A ) Signals of NUP1:GFP RE-ChIP-seq (20-kb window size), represented as the log 2 value of the ratio between normalized anti-GFP and IgG sequence coverage over Chromosome 1. For each tissue, the solid and dotted lines depict two replicates. ( B ) Distribution of NUP1-enriched domains across the genome viewed with the Integrative Genomics Viewer browser . ( C ) Percentage of NUP1-enriched genomic regions: (inf) inflorescence. ( D ) Venn diagram of genes enriched in four tissues.

Article Snippet: Next, the sheared chromatin was mixed with an equal volume of IP buffer (50 mM Hepes, pH 7.5; 150 mM NaCl; 5 mM MgCl 2 ; 10 μM ZnSO 4 ; 1% Triton X-100; 0.05% SDS) and then equally divided and incubated with anti-GFP antibody (Abcam, ab290) or normal rabbit IgG (Santa Cruz, sc-2027), respectively.

Techniques: Genome Wide, ChIP-sequencing, Sequencing

Journal: Nature Communications

Article Title: Light regulates stomatal development by modulating paracrine signaling from inner tissues

doi: 10.1038/s41467-021-23728-2

Figure Lengend Snippet: a , b Gene expression analyses of STOMAGEN in WT, hy5-215 , hy5-51 (a only), and HY5-OX by RT-qPCR. In ( a ), RNA was extracted from 3-day-old seedlings grown under the light. In ( b ), seedlings were grown in darkness for 4 days and were exposed to light for 0, 2, and 4 h before harvest. Values are mean + /− SEM, n = 3 biological replicates. One-way ( a ) or two-way ( b ) ANOVA with Tukey’s multiple comparisons test, P < 0.01. c Co-expression of HY5 and STOMAGEN in the mesophyll layer. Confocal analysis of 3-day-old abaxial cotyledons of a transgenic seedling harboring both HY5pro:HY5-YFP and STOMAGENpro:H2B-mScarlet-I . From left: YFP signals (yellow), mScarlet-I signals (magenta), autofluorescence (cyan) and merged image of all three channels. Scale bar, 50 μm. Three independent cotyledons were examined with similar results ( d ) Gene structure of STOMAGEN . Arrow indicates the translational start site. Vertical bars mark the position of a Z-box (upstream of TSS only). P1 to 3 represent region(s) tested by EMSA ( e ), DNA pull down ( f ) and ChIP-qPCR ( g ). e EMSA analysis showing the binding of HY5 to a promoter fragment of STOMAGEN (P2). Recombinant MBP (control) and MBP-HY5 were assayed for binding with the biotin-labeled P2 probe. An unlabeled probe (competitor) was used to determine binding specificity (lanes 4–6). f DNA pull-down analysis showing the binding of HY5 to P2 and its dependence on the Z-box. Biotin-labeled probes, including a P2 probe with a mutated Z-box (mP2), were used to pull-down recombinant MBP (control) and MBP-HY5. Results were analyzed by western blotting using an anti-MBP antibody. g ChIP-qPCR assays were performed on WT and HY5pro:HY5-YFP using an anti-GFP antibody. Seedlings were grown for 4 days in darkness before exposed to light for 4 h or kept in the dark. Promoter regions of STOMAGEN (see d) were tested. A genomic region downstream of STOMAGEN and IR1 (see “Methods”) were used as negative controls. Values are mean + /− SEM, n = 3 technical replicates. Assay was repeated with similar results. h GUS reporter assay of two independent lines of STOMAGENpro:GUS and mSTOMAGENpro:GUS , which carries a mutated Z-box in the P2 region, in WT and hy5-215 . Seedlings were grown for 3 days under the light. Scale bar, 2 mm. Light intensity used: 200 ( a ) or 100 µmol m −2 s −1 ( b , c , g , h ). The experiments in ( e ) and ( f ) were carried out two times with similar results.

Article Snippet: Total proteins were extracted from seedlings using 1× Laemmli sample buffer (Bio-Rad, 1610737) and immunoblotting was carried out with an HRP-conjugated anti-GFP antibody (Miltenyi Biotec, 130-091-83, 1:3000 dilution) or a Rabbit monoclonal anti-GFP antibody (Cell Signaling Technology, 2956, 1:1000 dilution) followed by anti-rabbit IgG-HRP (Cell Signaling Technology, 7074, 1:3000 dilution).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Transgenic Assay, ChIP-qPCR, Binding Assay, Recombinant, Control, Labeling, Western Blot, Reporter Assay

Journal: Neuron

Article Title: Nipbl Interacts with Zfp609 and the Integrator Complex to Regulate Cortical Neuron Migration

doi: 10.1016/j.neuron.2016.11.047

Figure Lengend Snippet: Zfp609 Is Expressed in Neural Progenitors and Regulates Cortical Neuron Migration (A) Composite bright field images of in situ hybridization on cortical cryosections at indicated stages of mouse development. Scale bar represents 200 μm. (B) Western blot with indicated antibodies on HEK293T lysates transiently transfected with wild-type or shRNA-resistant ( ∗ ) Zfp609-V5 expression constructs and control or Zfp609-targeting shRNA. Lamin B1 was used as a loading control. (C) Cryosections of mouse embryonic brains in utero electroporated with Zfp609 -targeting shRNAs and Zfp609 ∗ -V5 rescue construct, stained with GFP to visualize transfected cells. Ventricular (VZ), subventricular (SVZ), and intermediate zones (IZ) and cortical plate (CP) are indicated. Scale bar represents 100 μm. (D) Quantification of (C) showing percentage of GFP-expressing cells in indicated cortical regions. Error bars represent SEM, ∗ p < 0.05, ∗∗∗ p < 0.001; ns, non significant; two-tailed unpaired Student’s t test, n = 7. (E) Representative images showing morphology of electroporated neurons at E17.5 near the border between IZ and CP. Arrowheads point to multipolar cells; higher magnification in inset. Scale bar represents 20 μm. (F) Quantification of cell morphology in upper IZ. Error bars represent SEM, ∗ p < 0.05, two-tailed unpaired Student’s t test, n = 7 (control shRNA) and 8 ( Zfp609 shRNA). (G) Representative images of cryosections of electroporated mouse embryonic brains at postnatal day 2, stained with GFP antibody. Scale bar represents 100 μm. (H) Normalized expression levels in fragments per kilobase of exon per million mapped reads (FPKM) of Zfp608 and Zfp609 transcripts in NSCs. Western blot analysis of NSC lysate with Zfp609 antibody. Lamin B1 was used as a loading control. (I) Immunocytochemistry with V5 antibody on NSCs showing nuclear localization of ectopically expressed Zfp609-V5.

Article Snippet: Additional antibodies included V5 (R960-25, Invitrogen), Actin (A2066, Sigma), Vcp (ab11433, Abcam), and GFP (4745-1051, AbD Serotec).

Techniques: Migration, In Situ Hybridization, Western Blot, Transfection, shRNA, Expressing, Construct, Control, In Utero, Staining, Two Tailed Test, Immunocytochemistry

Journal: Neuron

Article Title: Nipbl Interacts with Zfp609 and the Integrator Complex to Regulate Cortical Neuron Migration

doi: 10.1016/j.neuron.2016.11.047

Figure Lengend Snippet: Nipbl Interacts with Zfp609 and Regulates Neuronal Migration (A) Colloidal Coomassie-stained SDS-PAA gel of Zfp609-V5 and control purification. Zfp609-V5 band is indicated by an arrow. Bands representing antibody heavy and light chain are indicated by an asterisk. (B) Western blot with V5 antibody on input, supernatant, and bound fractions of V5 affinity purification. (C) Western blot with indicated antibodies on V5 immunoprecipitates from Zfp609-V5-expressing NSCs. Benzonase (B) or ethidium bromide (EB) was added as indicated. Normal mouse IgG was used as control. (D) Western blot with indicated antibodies on Nipbl immunoprecipitates. Benzonase or ethidium bromide was added as indicated. Normal mouse IgG was used as control. (E) Western blot analysis with Nipbl antibody on GST pull-down fractions from NSC nuclear extract using GST-Zfp609 N-terminal (N), middle (M), and C-terminal (C) fragments or GST control. (F) Cryosections of mouse embryonic brains in utero electroporated with indicated Nipbl -targeting shRNAs, stained with GFP to visualize transfected cells. Ventricular (VZ), subventricular (SVZ), and intermediate zones (IZ) and cortical plate (CP) are indicated. Scale bar represents 100 μm. (G) Quantification of (F) showing percentage of GFP-expressing cells in indicated cortical regions. Error bars represent SEM, ∗ p < 0.05, ∗∗ p < 0.01, two-tailed unpaired Student’s t test, n = 4. (H) Quantification of cell morphology in upper IZ. Error bars represent SEM, ∗∗ p < 0.01, two-tailed unpaired Student’s t test, n = 4 (control, Nipbl shRNA 1) and 5 ( Nipbl shRNA 2). (I) Representative images of cryosections of electroporated mouse embryonic brains at postnatal day 2, stained with GFP antibody. Scale bar represents 100 μm.

Article Snippet: Additional antibodies included V5 (R960-25, Invitrogen), Actin (A2066, Sigma), Vcp (ab11433, Abcam), and GFP (4745-1051, AbD Serotec).

Techniques: Migration, Staining, Control, Purification, Western Blot, Affinity Purification, Expressing, In Utero, Transfection, Two Tailed Test, shRNA

Journal: Neuron

Article Title: Nipbl Interacts with Zfp609 and the Integrator Complex to Regulate Cortical Neuron Migration

doi: 10.1016/j.neuron.2016.11.047

Figure Lengend Snippet: Zfp609 and Nipbl Interact with Integrator to Regulate Cortical Migration (A) Western blot with indicated antibodies of V5 immunoprecipitates on Zfp609-V5-expressing or control NSC nuclear extract. (B) Immunoprecipitation of Ints1 analyzed by western blot with indicated antibodies. Benzonase (B) or ethidium bromide (EB) was added as indicated. Rabbit anti-GFP was used as control. (C) GST pull-down with Zfp609 N-terminal (N), middle (M), and C-terminal (C) fragments or GST control on NSC nuclear extract analyzed by western blot with indicated antibodies. (D) Heatmap of 7,030 active, 1,498 poised, 690 repressed, and 1,573 unmarked promoter proximal DHSs displaying 10 kb around DHS summits. Regions are ranked by normalized Zfp609 ChIP-seq signal, and mean ChIP-seq counts of indicated factors are plotted. (E) Heatmap of 3,912 active, 6,487 poised, 866 repressed, and 3,714 unmarked distal DHSs displaying 10 kb region around DHS summit. Regions are ranked by normalized Zfp609 ChIP-seq signal, and mean ChIP-seq counts of indicated factors are plotted. (F) Boxplot representing distribution of pausing indices of Zfp609 TSS-bound (n = 5,391) versus all other (n = 1,543) expressed (FPKM > 1) genes. Whiskers represent minimum and maximum values. p value by Mann-Whitney test is indicated. (G) Western blot on NSC lysates lentivirally transduced with the indicated shRNAs. Actin was used as a loading control. (H) Venn diagram displaying intersection of deregulated genes in Integrator KD with Zfp609/Nipbl target genes. (I) Normalized expression values from RNA-seq data on control or Ints1-depleted NSCs. Error bars represent SEM, ∗∗∗ p < 0.001, unpaired Student’s t test corrected for multiple comparisons using Holm-Sidak method, n = 3. (J) Cryosections of mouse embryonic brains in utero electroporated with Ints1 - and Ints11 (Cpsf3l) -targeting shRNAs, stained with GFP to visualize transfected cells. Ventricular (VZ), subventricular (SVZ), and intermediate zones (IZ) and cortical plate (CP) are indicated. Scale bar represents 100 μm. (K) Quantification of (J) showing percentage of GFP-expressing cells in indicated cortical regions. Error bars represent SEM, ∗∗ p < 0.01, two-tailed unpaired Student’s t test corrected for multiple comparisons using Holm-Sidak method, n = 4 (control) and 6 ( Ints1 and Ints11 shRNA).

Article Snippet: Additional antibodies included V5 (R960-25, Invitrogen), Actin (A2066, Sigma), Vcp (ab11433, Abcam), and GFP (4745-1051, AbD Serotec).

Techniques: Migration, Western Blot, Expressing, Control, Immunoprecipitation, ChIP-sequencing, MANN-WHITNEY, Transduction, RNA Sequencing, In Utero, Staining, Transfection, Two Tailed Test, shRNA