Journal: bioRxiv

Article Title: Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

doi: 10.64898/2026.01.18.700178

Figure Lengend Snippet: (a) The qExM framework targets endogenous proteins or protein complexes using pairs of antibodies against non-overlapping epitopes, leveraging western blot-effective antibodies enabled by expansion microscopy’s improved labeling efficiency. (b) Schematic representation of the labeling outcome categories in a qExM experiment. The total population of targets can be labeled by either antibody A (blue), antibody B (red), both antibodies (dual-labeled), or remain unlabeled. Each label provides a random subset of the total population, allowing assessment of the other label’s efficiency. (c) Mathematical formulation of the estimators used in qExM to calculate labeling efficiency of each antibody and the total abundance of target proteins, derived from the Chapman capture-recapture method. (d , e , f) Fluorescence microscopy images of U2OS cells prepared using three expansion microscopy protocols and collected and displayed with identical parameters: d: chemical fixation with glycidyl methacrylate anchoring (ChemGMA), e: cryo-fixation with formaldehyde/acrylamide anchoring (CryoFAA), and f: cryo-fixation with glycidyl methacrylate anchoring (CryoGMA). Cells were immunolabeled for Nup96-GFP (cyan), mitochondrial MT-CO1 (magenta), and α-tubulin (yellow). Scale bar 5 µm. (g) Signal to Noise measurements comparing the mitochondrial MT-CO1 signal across the three expansion microscopy protocols.

Article Snippet: In this study we used the following primary antibodies: for CI: NDUFS2 (Abcam ab192022), ND2 (Sigma MABS2047), for CIII: UQCRCP1 (Abcam ab14746), UQCRFS1/RISP (Abcam ab11925), for CIV: MT-CO1 (Abcam ab14705), MT-CO2 (Abcam ab79393), GFP (Abcam ab6673), Nup96 (ProteinTech-12329-1-AP), Nup54 (Sigma HPA035929), ELYS (Sigma HPA031658), P5CS (Proteintech 17719-1-AP, 68184-1-IG) All antibodies were used at a dilution of 1:100, except ELYS which was used at a concentration of 1:50.

Techniques: Western Blot, Labeling, Formulation, Derivative Assay, Fluorescence, Microscopy, Immunolabeling

Journal: bioRxiv

Article Title: Quantitative Expansion Microscopy for In Situ Estimation of Endogenous Target Abundance

doi: 10.64898/2026.01.18.700178

Figure Lengend Snippet: (a) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and Nup96 (blue) labeling. (b) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and Nup54 (blue) labeling. (c) qExM STED images of Nup96-mEGFP U2OS cells with mEGFP (red) and ELYS (blue) labeling. Smaller images in a-c show individual nuclear pores at higher magnification, demonstrating the characteristic ring-like arrangement of NPC subunits. (d) Structural model of the human nuclear pore complex highlighting the positions of Nup96-mEGFP (green), Nup54 (yellow), and ELYS (magenta) within the 8-fold symmetrical arrangement. (e) Image analysis workflow showing raw data (Image), segmentation mask (Segmentation), instance identification (Centers), and colocalization analysis (Colocalization). (f) Labeling efficiency estimates from qExM mEGFP and each target protein. “Direct Counting” refers to the labeling efficiency defined by the number of observed target per nuclear pore, knowing that there should be 8 total targets. No statistical significance was found between groups (mEGFP p = 0.23, Nup96 p= 0.65, Nup54 p= 0.28, ELYS p = 0.19) (g) Estimated abundance of protein subunits for each experiment, showing means of 7.4, 7.0, and 8.7 for Nup96, Nup54, and ELYS, respectively, closely matching the expected 8-fold symmetry. Scale bars: 500 nm (overview images) and 50 nm (insets). 3 experimental replicates were made for each imaging pair. 45 images of 1365 NPC wlabeled ith Nup96-mEGFP with Nup96. 41 images of 1465 NPC labeled with Nup96-mEGFP with Nup54. 32 images of 1118 NPC labeled with Nup96-mEGFP with ELYS.

Article Snippet: In this study we used the following primary antibodies: for CI: NDUFS2 (Abcam ab192022), ND2 (Sigma MABS2047), for CIII: UQCRCP1 (Abcam ab14746), UQCRFS1/RISP (Abcam ab11925), for CIV: MT-CO1 (Abcam ab14705), MT-CO2 (Abcam ab79393), GFP (Abcam ab6673), Nup96 (ProteinTech-12329-1-AP), Nup54 (Sigma HPA035929), ELYS (Sigma HPA031658), P5CS (Proteintech 17719-1-AP, 68184-1-IG) All antibodies were used at a dilution of 1:100, except ELYS which was used at a concentration of 1:50.

Techniques: Labeling, Imaging

Journal: PLoS Pathogens

Article Title: The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation

doi: 10.1371/journal.ppat.1009412

Figure Lengend Snippet: Antibodies Used in This Study.

Article Snippet: Nup98 , Atlas Antibodies , Cat# HPA074810.

Techniques:

Journal: bioRxiv

Article Title: CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

doi: 10.1101/630897

Figure Lengend Snippet: CNOT1 inhibits transcriptional activity of CLOCK-BMAL1 by interaction and stabilization of both CLOCK and BMAL1. (A) Effect of CNOT1 on transactivation by CLOCK-BMAL1 of a luciferase reporter gene driven by the mPer1 promoter. Data are means (±SD). N=3. A two tailed Student’s t -test was performed, and significant differences are represented by black asterisks. (***) P <0.001. (B) The carboxyl-terminus of CNOT1 is important for its inhibition on CLOCK-BMAL1. (C) Interactions were shown between CNOT1 and CLOCK or BMAL1 in HEK293T cells. (D) CNOT1 stabilized both CLOCK and BMAL1 in HEK293T cells. (E and F) Western blot analysis showed that CNOT1 increased stability of CLOCK (E) and BMAL1 (F) after addition of CHX (20 μg/ml). Densitometric analyses of the results from three independent experiments were shown below.

Article Snippet: Primary antibodies in this study were listed as followed: FLAG-HRP (Sigma, M2, catalogue No. A8592); HA-Tag (6E2) Mouse mAb (HRP Conjugate) (Cell Signaling, catalogue No. 2999); Phospho-(Ser/Thr) PKA Substrate Antibody (Cell Signaling, catalogue No. 9621); CNOT1 antibody (Sigma, HPA046577); pAb anti-BMAL1 (NOVUS, catalogue No. NB100-2288).

Techniques: Activity Assay, Luciferase, Two Tailed Test, Inhibition, Western Blot

Journal: bioRxiv

Article Title: CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

doi: 10.1101/630897

Figure Lengend Snippet: CNOT1 promotes phosphorylation and inhibits transcription of CLOCK-BMAL1. (A) Coexpressing CNOT1 induced phosphorylation of CLOCK and BMAL1 respectively. (B) cnot1 knockdown (KD) resulted in low phosphorylation levels and protein amounts of both CLOCK and BMAL1. (C) Densitometric analyses were shown of CNOT1, CLOCK and BMAL1 in control and cnot1 knockdown U2OS cells from three independent experiments. Data are means (±SD). N=3. A two tailed Student’s t -test was performed, and significant differences are represented by black asterisks. (**) P <0.01. (D and E) Ser to Ala combined mutations in CLOCK at 440, 441, 478, and 479 sites, or in BMAL1 at 42 and 246 sites resulted in compromised phosphorylation promoted by CNOT1 (D) and increased transactivation (E). (F) PKA-Cα inhibited transcriptional activity of CLOCK-BMAL1.

Article Snippet: Primary antibodies in this study were listed as followed: FLAG-HRP (Sigma, M2, catalogue No. A8592); HA-Tag (6E2) Mouse mAb (HRP Conjugate) (Cell Signaling, catalogue No. 2999); Phospho-(Ser/Thr) PKA Substrate Antibody (Cell Signaling, catalogue No. 9621); CNOT1 antibody (Sigma, HPA046577); pAb anti-BMAL1 (NOVUS, catalogue No. NB100-2288).

Techniques: Two Tailed Test, Activity Assay

Journal: bioRxiv

Article Title: CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

doi: 10.1101/630897

Figure Lengend Snippet: CNOT1 promotes associations between CLOCK or BMAL1 and endogenous PKA. (A) Interactions were shown between PKA and BMAL1, CNOT1, and CNOT1 increased the association between CLOCK and PKA in HEK293T cells in overexpressing conditions. (B) PKA-Cα could, but the PKA dead kinase (K73E and K169E) could not, phosphorylate both CLOCK and BMAL1. (C) Overexpressing Flag-tagged CLOCK, BMAL1 and CNOT1-C could pull down endogenous PKA, respectively. HEK293T cells were transfected by different plasmids and crude protein samples were pull-downed by Anti-Flag Affinity Gel. Negative control is mock transfection. The commercial PKA from the kit acts as a positive control. (D) CNOT1 promoted both CLOCK and BMAL1 binding to endogenous PKA. (E) Densitometric analyses were shown from three independent experiments. Data are means (±SD). N=3. A two tailed Student’s t -test was performed, and significant differences are represented by black asterisks. (*) P <0.05; (**) P <0.01.

Article Snippet: Primary antibodies in this study were listed as followed: FLAG-HRP (Sigma, M2, catalogue No. A8592); HA-Tag (6E2) Mouse mAb (HRP Conjugate) (Cell Signaling, catalogue No. 2999); Phospho-(Ser/Thr) PKA Substrate Antibody (Cell Signaling, catalogue No. 9621); CNOT1 antibody (Sigma, HPA046577); pAb anti-BMAL1 (NOVUS, catalogue No. NB100-2288).

Techniques: Transfection, Negative Control, Positive Control, Binding Assay, Two Tailed Test

Journal: bioRxiv

Article Title: CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

doi: 10.1101/630897

Figure Lengend Snippet: CNOT1 associates with CLOCK BMAL1 and promotes their phosphorylation by PKA in liver. (A) Anti-HA immuno-precipitation samples were shown on a SDS-PAGE gel. Lane1, protein markers; lane 2&3, controls from LAP/LAP mice liver; lane 4&5 from LAP/TETO::CLOCK-HA mice liver. (B) Mass spectrometry results identified a few endogenous proteins co-purified together with CLOCK-HA in liver. (C) Associations between CNOT1 and CLOCK were stronger at ZT8 than ZT16 in liver. (D) Accordingly, PKA phosphorylation levels of both CLOCK and BMAL1 were higher at ZT8 than ZT16 in liver.

Article Snippet: Primary antibodies in this study were listed as followed: FLAG-HRP (Sigma, M2, catalogue No. A8592); HA-Tag (6E2) Mouse mAb (HRP Conjugate) (Cell Signaling, catalogue No. 2999); Phospho-(Ser/Thr) PKA Substrate Antibody (Cell Signaling, catalogue No. 9621); CNOT1 antibody (Sigma, HPA046577); pAb anti-BMAL1 (NOVUS, catalogue No. NB100-2288).

Techniques: Immunoprecipitation, SDS Page, Mass Spectrometry, Purification

Journal: bioRxiv

Article Title: CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

doi: 10.1101/630897

Figure Lengend Snippet: Genetic deletion of PKA-Cα affects circadian cycles. (A) The target DNA location by CRISPR-Cas9 was shown in the PKA-Cα gene. (B) Sequencing results showed a 5-nt deletion preceding the PAM sequence. (C) The PKA-Cα mutant and the PKA overexpressing mutant respectively exhibited lower and higher PKA activity than that of WT. (D) The amplitudes and phases of both PKA overexpression and deletion mutants were altered in the circadian cycles. WT: wild type; OX: PKA overexpression; MT: PKA deletion mutant. (E) Period lengths were calculated based on four repeat experiments. (F) A schematic model of PKA and CNOT1 mediating phosphorylation and transcription of CLOCK-BMAL1 is shown. CLOCK and BMAL1 form a heterodimer, which is phosphorylated and activated by unknown kinases. Binding to the E-box leads to CLOCK-BMAL1 degradation and downstream clock controlled genes (CCG) transcription. CNOT1 bridges PKA and associates with CLOCK and BMAL1, increasing their phosphorylation and protein stability, in turn inhibiting their transactivation.

Article Snippet: Primary antibodies in this study were listed as followed: FLAG-HRP (Sigma, M2, catalogue No. A8592); HA-Tag (6E2) Mouse mAb (HRP Conjugate) (Cell Signaling, catalogue No. 2999); Phospho-(Ser/Thr) PKA Substrate Antibody (Cell Signaling, catalogue No. 9621); CNOT1 antibody (Sigma, HPA046577); pAb anti-BMAL1 (NOVUS, catalogue No. NB100-2288).

Techniques: CRISPR, Sequencing, Mutagenesis, Activity Assay, Over Expression, Binding Assay

Journal: Journal of innate immunity

Article Title: Viral attachment induces rapid recruitment of an innate immune sensor TRIM5? to the plasma membrane

doi: 10.1159/000346963

Figure Lengend Snippet: TRIM5αrh is found in higher buoyant density fractions upon virus-like particle (VLP) infection. A detergent-free method to separate higher buoyant density fractions were applied for all fractionation studies [44]. Representative images from 3 independent experiments are shown. (A) FRhK4 cells stably expressing HA-tagged TRIM5αrh protein (FRhK4T5αHA) treated with PBS (upper panel) or HIV-1 VLPs (lower panel) are shown. Flotillin-1 was used as a marker for flotation fractions, while Nup98 was used as a sedimenting control. Antibody against HIV-1 Capsid (CA) is shown, p24 is indicated by arrow. (B) Identical experiments were carried out as A, however using SIV VLPs or BSA-coated nanoparticles. (C) Identical fractionation experiments were carried out as A, however using wild-type FRhK4 proteins expressing endogenous levels of TRIM5αrh. Endogenous TRIM5αrh was probed using a commercially-available antibody against TRIM5 (ProSci Incorporated).

Article Snippet: Antibodies were used in the following concentrations: mouse anti-flotillin-1/reggie-2 (1:1000, BD Transduction Laboratories), mouse anti-flotillin-2/reggie-1 (1:1000, BD Transduction Laboratories), rat anti-HA (1:1000, Roche), rabbit anti-TRIM5 (1:200, ProSci Incorporated), mouse anti-p24 monoclonal antibodies, AG3.0 [ 45 ] and 183-H12-5C [ 46 ], and rabbit anti-Nup98 (1:500, a generous gift from Dr. Jan van Deursen).

Techniques: Infection, Fractionation, Stable Transfection, Expressing, Marker

Journal: Life Science Alliance

Article Title: Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect

doi: 10.26508/lsa.202201603

Figure Lengend Snippet: (A) Rif1–mKO2 cells (KYP1866 and KYP1867), in which the endogenous Rif1 was tagged with mKO2, harboring pREP41– Flag 3 vector (upper) or pREP41– Rif1– Flag 3 (lower) were grown in the absence of thiamine for 20 h, and were extracted by Triton X-100 and DNase I and remaining endogenous Rif1–mKO2 signals (mazenta) were observed. The nuclear envelope was stained with Nup98 antibody (green). (B, C) The numbers (B) and the intensities (C) of nuclear foci were quantified in Rif1–mKO2 cells harboring pREP41– Flag 3 (Vector) or pREP41– Rif1– Flag 3 (Rif1OE) grown as in (A). Source data are available for this figure.

Article Snippet: Nup98, a marker of the nuclear membrane, was detected with rat anti-Nup98 monoclonal antibody (1:500; Bioacademia) for 12 h at 4°C after blocking in PBS containing 3% BSA and 0.1% Tween 20.

Techniques: Plasmid Preparation, Staining

Journal: Life Science Alliance

Article Title: Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect

doi: 10.26508/lsa.202201603

Figure Lengend Snippet: (A, B, C) Rif1–mKO2 cells harboring vector ((A) and left panel of (C)) (KYP1866) or Rif1-overexpressing plasmid ((B) and right panel of (C)) (KYP1867) were pretreated with Triton X-100 and DNase I and stained with anti-Nup98 antibody (green; nuclear membrane) and Hoechst 33342 (blue; nuclei). The Hoechst signal is very low because of prior treatment with DNase I. In (A, B), mKO2 signals are in red, whereas they are in black in (C). Strong telomere signals of Rif1–mKO2 are detected in vector control, whereas multiple nuclear foci are detected upon overexpression of Rif1. This reflects its hetero-oligomerization with the overexpressed Rif1 protein and binding to chromosome arms. Source data are available for this figure.

Article Snippet: Nup98, a marker of the nuclear membrane, was detected with rat anti-Nup98 monoclonal antibody (1:500; Bioacademia) for 12 h at 4°C after blocking in PBS containing 3% BSA and 0.1% Tween 20.

Techniques: Plasmid Preparation, Staining, Membrane, Control, Over Expression, Binding Assay

Journal: Life Science Alliance

Article Title: Aberrant association of chromatin with nuclear periphery induced by Rif1 leads to mitotic defect

doi: 10.26508/lsa.202201603

Figure Lengend Snippet: Reagents & Resources.

Article Snippet: Nup98, a marker of the nuclear membrane, was detected with rat anti-Nup98 monoclonal antibody (1:500; Bioacademia) for 12 h at 4°C after blocking in PBS containing 3% BSA and 0.1% Tween 20.

Techniques: Recombinant, Protease Inhibitor, Membrane, Purification, Multiplex Assay, Software

Journal:

Article Title: Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes

doi: 10.1016/j.cell.2009.12.054

Figure Lengend Snippet: (A) Polytene chromosome spreads were stained with mAb414, anti-Sec13, anti-Nup88, anti-Nup98, anti-Nup154 and anti-gp210 as indicated; chromosomes are stained with DAPI (blue) here and throughout the paper, unless otherwise indicated. (B) Polytene chromosomes of third instar larvae of wandering LB stage from wt and Sec13 RNAi (top), Nup98 RNAi (middle) or nup8805043 homozygotes (bottom) were stained with anti-Sec13 and anti-Su(Hw), anti-Nup98 and a nti-Su(Hw), anti-Nup88 and anti-RNAP II, respectively. White arrows with numbers denote the same genomic locations for a particular Nup between chromosomes of wt and mutant larvae in a given panel. See also Figure S1.

Article Snippet: For ChIP-on-chip analysis, the Nup98 antibody- or normal rabbit IgG- immunoprecipitated DNA in duplicates was labeled and co-hybridized with input DNA to 2.1M Drosophila whole genome arrays by NimbleGen Systems, Inc. ChIP peak data were generated by NimbleGen, and genes with peaks of scaled log 2 -ratio ≥1 and FDR score ≤ 0.05 were selected as Nup98 targets.

Techniques: Staining, Mutagenesis

Journal:

Article Title: Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes

doi: 10.1016/j.cell.2009.12.054

Figure Lengend Snippet: (A) Chromosome spreads were co-stained with either anti-Sec13 (left) or anti-Nup88 (right) and RNAP II (MARA3 antibody that recognizes phosphorylated RNAP II). Proportional or non-proportional binding behavior of Sec13 is indicated with white and yellow arrows, respectively. Nup88 is found at sites devoid of RNAP II. (B) Chromosomes were co-stained with anti-Sec13 and mAb414, which shows recruitment of FG Nups to a subset of Sec13-positive sites, particularly at highly transcribing puffs. (C) Chromosomes were triple-stained with anti-Nup98, anti-Sec13 and anti-RNAP II (farred, color-coded as blue), showing co-localization of all 3 at multiple sites. (D) Co-localization analysis of a typical chromosomal arm, co-stained with either Nup88 and RNAP II (top plot) or with Nup98 and RNAP II (bottom plot), demonstrating lack of co-localization or co-localization, respectively. See also Figure S2.

Article Snippet: For ChIP-on-chip analysis, the Nup98 antibody- or normal rabbit IgG- immunoprecipitated DNA in duplicates was labeled and co-hybridized with input DNA to 2.1M Drosophila whole genome arrays by NimbleGen Systems, Inc. ChIP peak data were generated by NimbleGen, and genes with peaks of scaled log 2 -ratio ≥1 and FDR score ≤ 0.05 were selected as Nup98 targets.

Techniques: Staining, Binding Assay

Journal:

Article Title: Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes

doi: 10.1016/j.cell.2009.12.054

Figure Lengend Snippet: (A) Schematic illustration of the gene expression program of third instar larval stage, showing the expression profiles of Intermolt genes (red), Early genes (green), and the relative levels of ecdysone hormone (blue), with representative Puff Stages (PS) shown above. (B–C) Polytene chromosomes from larvae of different developmental stages (PS) were stained with anti-Sec13 and RNAP II; (B) during down-regulation of an Intermolt locus 68C, Sec13 levels are lost as RNAP II levels are decreased; (C) Developmental binding of Sec13 during up-regulation of Early loci 74EF and 75B, demonstrating presence of Sec13 either preceding or coinciding with appearance of phosphorylated RNAP II. (D) Polytene chromosomes from larvae of different developmental stages (PS) were stained with mAb414 and Sec13. Locus 62E is shown. (E) Isolated chromosomes were treated with buffer +/−RNase A and stained with anti-Pep, anti-Sec13, anti-Nup98 or mAb414. (F) Salivary glands were treated with buffer −/+FP and stained with control H5 antibody (anti-phosphoSer2 CTD of RNAP II), anti-Sec13, anti-Nup98 or mAb414. See also Figure S3.

Article Snippet: For ChIP-on-chip analysis, the Nup98 antibody- or normal rabbit IgG- immunoprecipitated DNA in duplicates was labeled and co-hybridized with input DNA to 2.1M Drosophila whole genome arrays by NimbleGen Systems, Inc. ChIP peak data were generated by NimbleGen, and genes with peaks of scaled log 2 -ratio ≥1 and FDR score ≤ 0.05 were selected as Nup98 targets.

Techniques: Expressing, Staining, Binding Assay, Isolation

Journal:

Article Title: Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes

doi: 10.1016/j.cell.2009.12.054

Figure Lengend Snippet: (A) Levels of phosphorylated RNAP II and of chromatin decondensation (puffing) of Early genes at 74EF and 75B (inset) are reduced in Sec13 RNAi knock-down larvae as compared to wt; and (B) in Nup98 RNAi knock-down larvae relative to Nup93 RNAi knock-down larvae. (C) Measured puff areas at 75B and 74EF were significantly reduced in Sec13 RNAi animals relative to wt, when normalized to the control non-puffing band. (D) RT-PCR analysis using primers for the Early gene transcripts E75A (75B) and E74A (74EF) in Sec13 RNAi and Nup98 RNAi relative to wt, Nup93 RNAi and Nup153 RNAi larvae. Ratios of mRNA levels of Early gene transcripts to control gene rp49 are shown. See also Figure S4.

Article Snippet: For ChIP-on-chip analysis, the Nup98 antibody- or normal rabbit IgG- immunoprecipitated DNA in duplicates was labeled and co-hybridized with input DNA to 2.1M Drosophila whole genome arrays by NimbleGen Systems, Inc. ChIP peak data were generated by NimbleGen, and genes with peaks of scaled log 2 -ratio ≥1 and FDR score ≤ 0.05 were selected as Nup98 targets.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes

doi: 10.1016/j.cell.2009.12.054

Figure Lengend Snippet: (A) Chromosomes were stained with RNAPII and Sec13 right after heat shock (HS) or after 20 min recovery (HS + recovery). Yellow arrows point to sites of bound Sec13 that do not correspond to the 9 known heat shock loci (marked by RNAPII at the HS time point). (B) Chromosomes of wt or Sec13 RNAi larvae were stained with RNAP II and Sec13 at the HS + recovery time point. RNAP II accumulation at the recovering transcription sites, observed in wt, is not seen in the Sec13 RNAi larvae. (C) Genome-wide levels of active RNAP II were compared between HS and HS + recovery time points for wt, Sec13 RNAi and Nup98 RNAi larvae. (D) Genome-wide levels of Nup98 recruited during heat shock recovery are shown to be reduced in Sec13 RNAi larvae relative to wt. See also Figure S5.

Article Snippet: For ChIP-on-chip analysis, the Nup98 antibody- or normal rabbit IgG- immunoprecipitated DNA in duplicates was labeled and co-hybridized with input DNA to 2.1M Drosophila whole genome arrays by NimbleGen Systems, Inc. ChIP peak data were generated by NimbleGen, and genes with peaks of scaled log 2 -ratio ≥1 and FDR score ≤ 0.05 were selected as Nup98 targets.

Techniques: Staining, Genome Wide

Journal:

Article Title: Chromatin-bound nuclear pore components regulate gene expression in higher eukaryotes

doi: 10.1016/j.cell.2009.12.054

Figure Lengend Snippet: (A) A representative example of Nup98 target sites obtained from ChIP-on-chip analysis, plotted relative to Lamin binding sites and annotated genes. (B) Distribution of Nup98 target and non-target genes relative to expression levels. (C) ChIP analysis of candidate target genes of Sec13 and Nup98 (CG6014/78D, Hph/82EF, CG13800/62E) and of control neighboring genes in Drosophila S2 cells, showing enrichment of target genes in fractions immunoprecipitated by Nup antisera (blue and red bars, respectively) as percentage of input DNA, with levels immunoprecipitated by the normal rabbit IgG subtracted. (D) Fold decrease in mRNA levels of Nup target genes in S2 cells treated with dsRNA against Sec13 or Nup98, relative to S2 cells treated with control dsRNA, as detected by microarray analysis. See also Figures S6 and S7.

Article Snippet: For ChIP-on-chip analysis, the Nup98 antibody- or normal rabbit IgG- immunoprecipitated DNA in duplicates was labeled and co-hybridized with input DNA to 2.1M Drosophila whole genome arrays by NimbleGen Systems, Inc. ChIP peak data were generated by NimbleGen, and genes with peaks of scaled log 2 -ratio ≥1 and FDR score ≤ 0.05 were selected as Nup98 targets.

Techniques: Binding Assay, Expressing, Immunoprecipitation, Microarray