Journal:

Article Title: Extracellular Signal-Regulated Kinase Activates Topoisomerase II? through a Mechanism Independent of Phosphorylation

doi:

Figure Lengend Snippet: Coimmunoprecipitation of endogenous topoisomerase IIα and ERK2 from nuclear extracts. ERK2 was immunoprecipitated from nuclear extracts prepared from nocodazole-treated NIH 3T3 cells; this was followed by immunoblotting to visualize both ERK2 and coimmunoprecipitating topoisomerase (Topo) IIα. Lanes 3 and 4 show increasing amounts of immunoprecipitated ERK2 and coimmunoprecipitated topoisomerase IIα. A small amount of topoisomerase IIα nonspecifically bound to the protein A-(Prot. A)-Sepharose resin (lane 2). Ten percent of the extract volume used for the immunoprecipitations served as a loading control (lane 1).

Article Snippet: For coimmunoprecipitations of endogenous ERK2 and topoisomerase IIα, nuclear extracts from nocodazole-treated NIH 3T3 cells were incubated with 0, 0.2, or 2 μg of anti-ERK2 antibody (C-14; Santa Cruz Biotechnology) for 2 h on ice; this was followed by addition of 20 μl of protein A-Sepharose (Pharmacia) that had been pretreated with BSA at 0.5 mg/ml.

Techniques: Immunoprecipitation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Effect of Neddylation Inhibition on Inflammation-Induced MMP9 Gene Expression in Esophageal Squamous Cell Carcinoma

doi: 10.3390/ijms22041716

Figure Lengend Snippet: Effect of tumor necrosis factor-alpha (TNF-α) and MLN4924 on the signaling pathways mediating matrix metalloproteinase 9 (MMP9) gene expression in esophageal squamous cell carcinoma (ESCC) cells. ( A ) Proteome profiling of the nuclear factor kappa B (NFκB) pathway by antibody array analyses in the KYSE150 cells treated with MLN4924 and TNF-α. Protein lysates from the untreated controls and cells treated with MLN4924 or TNF-α alone or in combination were analyzed using a human NFκB array (R&D). ( B ) Bar graphs showing the phosphorylation ratio (upper) and the protein level ratio (bottom) calculated after the semi-quantitative analysis of selected proteins. Results are presented as means ± SEM from duplicates. * p < 0.001, ** p < 0.05 vs. controls. The complete array is shown in . ( C ) Western blot analysis showing time-dependent activation of inhibitor of nuclear factor kappa B-alpha (IκB-α), NFκB/p65 and c-Jun in the KYSE150 cells treated with TNF-α (30 ng/mL). ( D ) MLN4924-dependent changes in the activation of IκB-α, NFκB/p65 and c-Jun as well as increasing levels of cyclin dependent kinase inhibitor 1A (CDKN1a/p21) protein in the KYSE150 cells within 24 h. ( E ) A dose-dependent effect of MLN4924 on activation of NFκB/p65 and c-Jun signaling pathways in the KYSE150 cells treated with TNF-α (30 g/mL) for 24 h. The effect of the KYSE70 cells treatment with different concentrations (0.25, 0.5, 1.0, 2.5 and 5.0 µM) of MLN4924 for 24 and 48 h is shown in .

Article Snippet: To determine the relative levels of 41 total and 4 serine/tyrosine phosphorylated proteins involved in NFκB signal transduction, the Proteome Profiler Human NF-κB Pathway Array (R&D Systems, ARY029) was used.

Techniques: Protein-Protein interactions, Gene Expression, Ab Array, Phospho-proteomics, Western Blot, Activation Assay

Journal: Scientific Reports

Article Title: Ethanol and C2 ceramide activate fatty acid oxidation in human hepatoma cells

doi: 10.1038/s41598-018-31025-0

Figure Lengend Snippet: PPARα antagonist GW6471 restores ethanol-mediated inhibition of FA oxidation and increased PLIN2 expression in C2 ceramide treated cells. ( A–F ) VL-17A cells were treated for 48 h with control or ethanol (100 mM)-containing media supplemented with 10 µM C2 ceramide. ( A–C ) cells were treated with 5 µM GW6471 for 24 h. ( A ) Oleate oxidation was quantified by measuring 3 H water liberation from 3 H labelled oleate (N = 5). ( B ) Densitometric quantification of PLIN2 and GAPDH measured in cell lysates by western blot (N = 3). ( C ) PPARα binding in nuclear extracts were quantified by transcription factor binding assay. ( D–F ) cells were transfected with scramble or PPARα siRNA 5 days before ethanol treatment. ( D ) PPARα mRNA was assayed by real time RT-PCR. ( E ) Firefly and renilla luciferase activity was assayed in cell extracts 48 h following transfection with the Cignal PPAR Reporter plasmid. ( F ) Oleate oxidation was quantified as in A. Data presented as mean +/−SEM. *p < 0.05 relative to control, ***p < 0.01 relative to control.

Article Snippet: PPARα DNA-binding in 7 µg of nuclear protein was measured using the Transcription Factor Assay Kit (Cayman Chemical) according to manufacturer’s instructions.

Techniques: Inhibition, Expressing, Western Blot, Binding Assay, Transfection, Quantitative RT-PCR, Luciferase, Activity Assay, Plasmid Preparation

Journal: The Journal of Cell Biology

Article Title: Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, β-catenin, and the phosphatase DEP-1/CD148

doi: 10.1083/jcb.200209019

Figure Lengend Snippet: VEGFR-2 association and dephosphorylation requires the β -catenin binding domain of VE-cadherin. VEC-null cells were transfected with VE-cadherin wild type or truncated mutants lacking β-catenin (Δ-βcat) or p120 (Δ-p120) binding domains. Intra, intracellular region; extra, extracellular region (C). (A) After stimulation with VEGF (80 ng/ml) for 5 and 30 min, cell extracts were immunoprecipitated (IP) with antibodies to VEGFR-2 (αVEGFR-2) and immunoblotted (IB) with antibodies to phosphotyrosine (αphosphoTyr), VEGFR-2 (αVEGFR-2), and VE-cadherin (αVEC). Wild-type (molecular mass, ∼120 kD) and Δ-p120 VE-cadherin (molecular mass, ∼100 kD) were coimmunoprecipitated with VEGFR-2 (A, lower panel). Receptor phosphorylation was significantly reduced in VEC-positive and Δ-p120, but not in Δ-βcat, in comparison with VEC-null cells. The quantification of receptor phosphorylation data from three experiments ± SD is shown in A on the right. The values represent the ratio between the phosphorylated and total amount of VEGFR-2 and are normalized to the ratio calculated in untreated VEC-positive cells. The peak of VEGFR-2 phosphorylation at 5 min is similar in VEC-null and Δ-βcat, but lower in Δ-p120. At longer stimulation (30 min), the level of phosphorylation of VEGFR-2 in Δ-p120 was comparable to VEC-positive cells. Incubation of VEC-positive cell extract with nonimmune (NI) rabbit immunoglobulin (matched with VEGFR-2 antibody used for IP) did not precipitate bands corresponding to either VEGFR-2 or VE-cadherin, last lane from the left (IP NI). (B) VE-cadherin mutants modulate endothelial growth induced by VEGF. VEC-null and Δ-βcat had comparable effects and were the most permissive mutations in terms of cell proliferation (>160% increase over VEC-positive cells). Mutations that affected binding of p120 (Δ-p120) allowed cell proliferation, but to a lower extent (60% increase over stimulation of VEC-positive cells). Proliferation was measured as BrdU incorporation as described in the legend to . Mean ± SD of three independent experiments, each in duplicate, is shown.

Article Snippet: BrdU incorporation into nuclear structures was put in evidence with anti-BrdU antibodies (mouse monoclonal, Amersham Biosciences), followed by TRITC-conjugated antibody to mouse immunoglobulin (DakoCytomation) in the presence of Hoechst 33258 (0.1 μg/ml).

Techniques: De-Phosphorylation Assay, Binding Assay, Transfection, Immunoprecipitation, Incubation, BrdU Incorporation Assay

Journal: The Journal of Cell Biology

Article Title: Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, β-catenin, and the phosphatase DEP-1/CD148

doi: 10.1083/jcb.200209019

Figure Lengend Snippet: The absence of β -catenin enhances VEGF-induced phosphorylation of VEGFR-2 and cell proliferation. Endothelial cells derived from β-catenin–null embryos (β-cat null) did not express β-catenin in comparison with cells obtained from β-catenin–positive (β-cat positive) littermate animals. (A) By immunofluorescence analysis, VE-cadherin was expressed at a comparable level and was concentrated at cell–cell contacts in both β-catenin–null and –positive cells. Cell junctions were negative for β-catenin in null cells. Bars: (phase contrast) 100 μm; (VE-cadherin and β-catenin) 20 μm. (B) Immunoprecipitation (IP) of cell extracts with VE-cadherin antibodies followed by Western blot (IB) with anti–VE-cadherin (αVE-cadherin) or β-catenin (αβ-catenin) antibodies showed absence of the last protein in the complex. (C) The absence of β-catenin enhanced the extent and duration of VEGFR-2 phosphorylation in response to VEGF (80 ng/ml). IP with anti–VEGFR-2 and Western blot with antiphosphotyrosine and anti–VEGFR-2 antibodies. (D) VE-cadherin could be coimmunoprecipitated with VEGFR-2 only in β-positive cells after VEGF (80 ng/ml for 5 min). IP with anti–VEGFR-2 and Western blot with anti–VEGFR-2 and anti–VE-cadherin antibodies. (E) Confluent β-cat–null endothelial cells incorporated BrdU 2–2.5-fold more than β-cat–positive cells in response to stimulation with VEGF (80 ng/ml for 24 h). Incorporation of BrdU was measured and calculated (mean of three independent experiments ± SD) as in . Two independent pairs of both β-positive and β-null endothelial cells obtained from littermate embryos of different mothers have been tested with comparable results.

Article Snippet: BrdU incorporation into nuclear structures was put in evidence with anti-BrdU antibodies (mouse monoclonal, Amersham Biosciences), followed by TRITC-conjugated antibody to mouse immunoglobulin (DakoCytomation) in the presence of Hoechst 33258 (0.1 μg/ml).

Techniques: Derivative Assay, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 is upregulated by NF‐κB and YY1. (A) The predicted NF‐κB and YY1 binding sites on LINC01578 promoter. NF‐κB and YY1 bind sites were at −438 and −4 positions relative to the transcription start site, respectively. (B) ChIP assays were performed in DLD‐1 cells to measure the binding of NF‐κB and YY1 on LINC01578 promoter. A distant region without NF‐κB and YY1 binding sites was used as NC (P3). (C) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with PBS or 10 ng·mL −1 TNF‐α for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with PBS or 10 ng·mL −1 TNF‐α for 24 h were detected by western blot. (D) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with DMSO or 5 µ m JSH‐23 for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with DMSO or 5 µ m JSH‐23 for 24 h were detected by western blot. (E) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and p65 overexpression vector. p65 overexpression efficiencies were detected by western blot. (F) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against p65. p65 knockdown efficiencies were detected by western blot. (G) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and YY1 overexpression vector. YY1 overexpression efficiencies were detected by western blot. (H) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against YY1. YY1 knockdown efficiencies were detected by western blot. (I) LINC01578 expression in DLD‐1 cells treated with PBS or 10 ng·mL −1 TNF‐α for 24 h. (J) LINC01578 expression in DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 for 24 h. (K) LINC01578 expression in DLD‐1 cells transfected with p65 overexpression vector. (L) LINC01578 expression in DLD‐1 cells transfected with siRNAs against p65. (M) LINC01578 expression in DLD‐1 cells transfected with YY1 overexpression vector. (N) LINC01578 expression in DLD‐1 cells transfected with siRNAs against YY1. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant, by one‐way ANOVA followed by Dunnett's multiple comparisons test (B) or Student's t ‐test (C‐N).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Binding Assay, Luciferase, Transfection, Western Blot, Over Expression, Plasmid Preparation, Expressing

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 activates NF‐κB signaling. (A) p50 IHC staining of liver metastatic tumors derived from LINC01578‐overexpressed and control DLD‐1 cells. Scale bars, 50 µm. (B) p50 IHC staining of liver metastatic tumors derived from LINC01578‐depleted and control LoVo cells. Scale bars, 50 µm. (C) Nuclear p50 and p65 levels in LINC01578‐overexpressed and control DLD‐1 cells were detected by western blot. (D) Nuclear p50 and p65 levels in LINC01578‐depleted and control LoVo cells were detected by western blot. (E) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐overexpressed and control DLD‐1 cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (F) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐depleted and control LoVo cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (G) p50 and p65 activation in nuclear extracts from LINC01578‐overexpressed and control DLD‐1 cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. (H) p50 and p65 activation in nuclear extracts from LINC01578‐depleted and control LoVo cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. Data are shown as mean ± SD based on n = 6 mice in each group (A,B) or three independent experiments (C–H). * P < 0.05, ** P < 0.01 by the Mann–Whitney test (A), Kruskal–Wallis test followed by Dunn's multiple comparisons test (B), or one‐way ANOVA followed by Dunnett's multiple comparisons test (E–H).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Immunohistochemistry, Derivative Assay, Western Blot, Luciferase, Binding Assay, Transfection, Activity Assay, Activation Assay, Transcription Factor Assay, MANN-WHITNEY

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: Blocking of NF‐κB signaling abolished the oncogenic roles of LINC01578 in colon cancer. (A) Cell viability of LINC01578 overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by Glo cell viability assay. (B) Cell proliferation of LINC01578‐overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by EdU assays. Scale bars, 100 µm. (C) Cell migration of LINC01578‐overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by transwell migration assays. Scale bars, 100 µm. (D) Cell invasion of LINC01578‐overexpressed and control DLD‐1 cells treated with DMSO or 5 µ m JSH‐23 was determined by transwell invasion assays. Scale bars, 100 µm. Data are shown as mean ± SD based on three independent experiments. ** P < 0.01, ns, not significant, by Student's t ‐test.

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Blocking Assay, Viability Assay, Migration

Journal: Molecular Oncology

Article Title: Long noncoding RNA LINC01578 drives colon cancer metastasis through a positive feedback loop with the NF‐κB/YY1 axis

doi: 10.1002/1878-0261.12819

Figure Lengend Snippet: LINC01578 forms a positive feedback loop with NF‐κB/YY1. (A) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector were determined by qRT‐PCR and western blot. (B) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 were determined by qRT‐PCR and western blot. (C) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector and treated with 5 µ m JSH‐23 were determined by qRT‐PCR and western blot. (D) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 and treated with 5 µ m JSH‐23 were determined by qRT‐PCR and western blot. (E) A schematic model of positive feedback loop between LINC01578 and NF‐κB/YY1. (F) Luciferase reporter assays for LINC01578‐overexpressed and control DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (G) Luciferase reporter assays for LINC01578‐depleted and control LoVo cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (H) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (I) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. (J) ChIP assays using YY1 antibody were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (K) ChIP assays using YY1 antibody were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. Data are shown as mean ± SD based on three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, ns, not significant, by Student's t ‐test (A,C,F,H,J), or one‐way ANOVA followed by Dunnett's multiple comparisons test (B,D,G,I,K).

Article Snippet: The nuclear extracts from indicated cells were used to measure NF‐κB transcription factor DNA binding activities using the NF‐κB p50 Transcription Factor Assay Kit (Cat. ab207217; Abcam) and NF‐κB p65 Transcription Factor Assay Kit (Cat. ab133112; Abcam) following the manufacturer's protocols.

Techniques: Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Infection, Luciferase

Journal: Scientific Reports

Article Title: Resveratrol Ameliorates Mitophagy Disturbance and Improves Cardiac Pathophysiology of Dystrophin-deficient mdx Mice

doi: 10.1038/s41598-018-33930-w

Figure Lengend Snippet: Levels of mtDNA with deletion and tissue ROS were increased in the mdx mouse heart. ( A ) Schematic depicting the regions of the mouse mitochondrial genome (mtDNA) amplified by long-range PCR [nucleotide positions (np) 9984-3577 and np 3553–9990] and the qPCR methods. ( B ) mtDNA content determined by qPCR amplifying the D-loop and COX2 regions and nuclear RPS18 genome region. N = 4. ( C ) Representative gel images of long-range PCR of myocardial DNA samples. For quantification, the results of 10, 5, and 2.5 ng of DNA from an intact mouse heart per reaction were included. The nuclear Gapdh gene was amplified as an internal control. ( D ) Levels of long-range PCR products normalized to Gapdh . N = 4. ( E ) Representative Immunoblots for VDAC1, SDHA, Rieske, HSP60, and GAPDH. ( F ) Levels of mitochondrial proteins in the hearts. ( G ) Dihydroethidium (DHE) fluorescence (red) images in heart sections from 22-week-old control and mdx mice. ( H ) Relative DHE fluorescence intensity. Eight images randomly captured from 4 hearts were analyzed in each group. ( I ) qPCR analyses of Nppa and Nppb genes normalized to β-actin. N = 4. All data were analyzed by unpaired 2-tailed Student’s t test. *P < 0.05. NS: not significant. a.u.: arbitrary units. kb: kilobase.

Article Snippet: To amplify mtDNA and nuclear DNA, StepOne real-time PCR system (Thermo Fisher Scientific) and GoTaq qPCR Master Mix (Promega, A6001) were used.

Techniques: Amplification, Long Range PCR, Control, Western Blot, Fluorescence

Journal: Cell Death & Disease

Article Title: Aldolase B-driven lactagenesis and CEACAM6 activation promote cell renewal and chemoresistance in colorectal cancer through the Warburg effect

doi: 10.1038/s41419-023-06187-z

Figure Lengend Snippet: A Metabolomic analysis based on nuclear magnetic resonance (NMR) showing metabolite levels in CRC cells with or without ALDOB overexpression. Black dashed lines indicate relative metabolite levels in control-treated cells. B Lactate levels in medium from cells with control or ALDOB overexpression. C Transwell-based co-culture system for assessment of cell growth (left panel) and chemoresistance (right panels). D Supplementation of medium from cells with or without ALDOB overexpression for assessment of cell proliferation (left panel) and chemoresistance (right panels). E Western blots demonstrating levels of the indicated proteins in cells with or without ALDOB overexpression. The statistical analysis is displayed in the right panel. F Immunofluorescence assay showing the expression of exogenous ALDOB (ALDOB-MYC) and LDHB in CRC cells transfected with ALDOB expression plasmid. The scale bar represents 20 μm. G Western blots showing the levels of the indicated proteins in cells treated with different concentrations of lactate. H Immunofluorescence assay showing LDHB expression in CRC cells treated with indicated concentrations of lactate. The scale bar represents 20 μm. All P values were obtained using the paired two-tailed Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: A mouse monoclonal antibody targeting the MYC tag (Proteintech, Cat. 60003-2-Ig), and a rabbit monoclonal antibody to LDHB (Proteintech, Cat. 14824-1-AP) were used at a 1:200 dilution.

Techniques: Nuclear Magnetic Resonance, Over Expression, Control, Co-Culture Assay, Western Blot, Immunofluorescence, Expressing, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 3. hnRNP H/F proteins bind exonic regulatory elements in exon 18b. (A, left) Diagram of wild-type and SWAP constructs in which the positions of exon 18a and exon 18b are switched. (Right) 32P RT–PCR assays of RNAs extracted from transfected HeLa cells using T7 primer and Ex19R. PCR products were digested with PstI. (B, left) Diagram of point-swapping mutant constructs. The details of a series of mutants are described in Supplemental Figure S4. (Right) 32P RT–PCR assays of RNAs extracted from transfected HeLa cells using T7 primer and Ex19R. PCR products were digested with PstI or HaeII. The E12 (exon18a inclusion) percentage was calculated by dividing exon 18a signal by the sum of exon 18a and 18b signals and is indicated below. (C) 32P RT–PCR assays of additional point-swapping mu- tants in sections 3 and 6 in HeLa cells. (D, top) Sequence comparison of sections 3-2 and 6-2. The putative hnRNP H/F-binding sites are underlined. The RNA affinity assay used biotinylated 3-2 and 6-2 short RNA oligonucleotides. Isolated proteins were analyzed by Western blot using the indicated antibodies.

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Construct, Reverse Transcription Polymerase Chain Reaction, Transfection, Mutagenesis, Sequencing, Comparison, Binding Assay, Isolation, Western Blot

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 4. hnRNP H/F expression and recruitment to exonic regulatory elements decrease during differentiation. (A) E12/E47 ratio was examined by 32P RT–PCR. PCR products were digested by PstI. (B) Expression of pluripotency and differentiation markers as determined by quantitative PCR (qPCR) after 8 d of differentiation. The P-value was determined by two-tailed Student’s t-test. (∗) P < 0.05. (C) CLIP analysis to measure the hnRNP H protein enrichment on exon 18b of TCF3 mRNA by qPCR after 8 d of differentiation. Data were nor- malized to hnRNP H immunoprecipitation signal in undifferentiated H9 cells. All results represent the means ± SD from at least three independent experiments. (∗) P < 0.05. (D) Expression of hnRNP H and hnRNP F during 8 d of differentiation of H9 cells. mRNA levels were determined by qPCR. (E) Protein levels were analyzed by Western blot using the indicated antibodies (left), and the bar graph shows its quantification (right).

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Two Tailed Test, Protein Enrichment, Immunoprecipitation, Western Blot

Journal: Genes & development

Article Title: TCF3 alternative splicing controlled by hnRNP H/F regulates E-cadherin expression and hESC pluripotency.

doi: 10.1101/gad.316984.118

Figure Lengend Snippet: Figure 5. hnRNP H1 and hnRNP F regulate TCF3 splicing and hESC morphology. (A) E12/E47 ratio in hESCs after the indicated siRNA treatment analyzed by 32P RT–PCR. RT–PCR products were digested by PstI and resolved by 5% PAGE. (Top) The E12 percentage is in- dicated below. (Bottom) Immunoblots showing protein levels after the indicated siRNA treatment. (B) E12/E47 ratio in 293T cells after the indicated siRNA treatment analyzed by 32P RT–PCR. (C) Splicing assays analyzed by 32P RT–PCR using T7 primer and Ex19R. The TCF3 minigene and the indicated expression plasmids were transfected into 293T cells. Total RNA from the transfected cells was used for 32P RT–PCR. (D) Cell morphology of H9 hESCs transfected by the indicated siRNAs. (E) Expression of pluripotency markers in hnRNP H/F knockdown cells, determined by qPCR. (F) Expression of differentiation markers in H9 hESCs after the indicated siRNA treatment, determined by qPCR.

Article Snippet: Western blotting Cells were lysed with SDS sample buffer (50 mM Tris at pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxy- cholate, 1× proteinase inhibitor cocktail [Biotools]), resolved by SDS-PAGE, transferred to nitrocellulose membranes, and incubated with primary antibodies detecting CDH1 (1:1000; Cell Signaling), hnRNP H1 (1:1000; Bethyl Laboratories), hnRNP F (1:1500; Santa Cruz Biotechnology), GAPDH (1:20,000; Sigma), or ACTB (1:30,000; Sigma) overnight at 4°C.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection, Knockdown