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Santa Cruz Biotechnology protease inhibitor mixture
Protease Inhibitor Mixture, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/protease inhibitor mixture/product/Santa Cruz Biotechnology
Average 92 stars, based on 22 article reviews
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protease inhibitor mixture - by Bioz Stars, 2020-11
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Sonication:

Article Title: Defective Sphingosine-1-phosphate metabolism is a druggable target in Huntington’s disease
Article Snippet: .. Total lysate preparation Tissues were homogenized in lysis buffer containing 20 mMTris, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 20 mMNaF, 2 mM Na3 V04 , and protease inhibitor mixture (Santa Cruz, Cat N. sc-29131) and sonicated with 2 × 10 s pulses. ..

Chromatography:

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *
Article Snippet: .. For purification of mouse PECAM, a microsome fraction (∼50 mg of protein) was prepared by ultracentrifugation of mouse lung homogenates, solubilized in 1% Triton X-100 in TBS containing a protease inhibitor mixture, diluted with TBS until the final concentration of Triton X-100 was 0.3%, and subjected to immunoaffinity chromatography in which a de- N -glycosylated anti-PECAM antibody (200 μg; M-20; Santa Cruz Biotechnology) by peptide N -glycosidase F was coupled to 60 mg of tosylactivated Dynabeads M-280 (Invitrogen). .. After non-specifically bound proteins were eluted with a non-related peptide solution (100 μg/ml), the PECAM immunocomplex was digested with trypsin and chymotrypsin (Nacalai Tesque) in 50 m m ammonium bicarbonate at 37 °C for 16 h, boiled for 10 min, and treated with 30 μl of peptide N -glycosidase F at 37 °C for 16 h. N -Glycans released from 5 μg of pure PECAM were enriched with Oasis HLB Extraction Cartridges (Waters) and pyridylaminated as described previously ( , ).

Protease Inhibitor:

Article Title: MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch-Nyhan disease?
Article Snippet: .. Cells were cultured, lysed in six-well plates and the extracts were obtained using RIPA buffer with a protease inhibitor mixture (Santa Cruz, Inc.) (50 m m Tris–Cl, pH 8, 150 m m NaCl, 0.5% of deoxycholate, 0.1% SDS, 2 m m EDTA, 1% aprotinin, 1 m m PMSF, 1 m m sodium orthovanadate). ..

Article Title: Transforming Growth Factor-?1 (TGF-?1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts *
Article Snippet: .. Cells were grown to confluence in 35-mm dishes, and total cellular protein was extracted in radioimmune precipitation assay lysis buffer containing 1% protease inhibitor mixture, 1% PMSF, and 1% sodium orthovanadate (Santa Cruz Biotechnology, Inc.), as described previously ( ). .. Cell protein samples were incubated with anti-EGFR antibody-conjugated Magnabind beads (Thermo Scientific Pierce) overnight at 4 °C.

Article Title: Peptidomimetic blockade of MYB in acute myeloid leukemia
Article Snippet: .. Cells were lysed in RIPA buffer (Thermo Fisher) supplemented with a protease inhibitor mix comprised of AEBSF (0.5 mM concentration, Santa Cruz, SC-202041B), Bestatin (0.01 mM, Fisher/Alfa Aesar, J61106-MD), Leupeptin (0.1 mM, Santa Cruz, SC-295358B), and Pepstatin (0.001 mM, Santa Cruz, SC-45036A). .. Lysates were mechanically disrupted using Covaris S220 adaptive focused sonicator, according to the manufacturer’s instructions (Covaris, Woburn, CA).

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *
Article Snippet: .. For purification of mouse PECAM, a microsome fraction (∼50 mg of protein) was prepared by ultracentrifugation of mouse lung homogenates, solubilized in 1% Triton X-100 in TBS containing a protease inhibitor mixture, diluted with TBS until the final concentration of Triton X-100 was 0.3%, and subjected to immunoaffinity chromatography in which a de- N -glycosylated anti-PECAM antibody (200 μg; M-20; Santa Cruz Biotechnology) by peptide N -glycosidase F was coupled to 60 mg of tosylactivated Dynabeads M-280 (Invitrogen). .. After non-specifically bound proteins were eluted with a non-related peptide solution (100 μg/ml), the PECAM immunocomplex was digested with trypsin and chymotrypsin (Nacalai Tesque) in 50 m m ammonium bicarbonate at 37 °C for 16 h, boiled for 10 min, and treated with 30 μl of peptide N -glycosidase F at 37 °C for 16 h. N -Glycans released from 5 μg of pure PECAM were enriched with Oasis HLB Extraction Cartridges (Waters) and pyridylaminated as described previously ( , ).

Article Title: Hyaluronan Orchestrates Transforming Growth Factor-β1-dependent Maintenance of Myofibroblast Phenotype
Article Snippet: .. Cells were then lysed using 1% protease inhibitor mixture, 1% phenymethylsulfonyl fluoride, and 1% sodium orthovanadate in RIPA lysis buffer (Santa Cruz Biotechnology, Inc.). ..

Article Title: Defective Sphingosine-1-phosphate metabolism is a druggable target in Huntington’s disease
Article Snippet: .. Total lysate preparation Tissues were homogenized in lysis buffer containing 20 mMTris, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 20 mMNaF, 2 mM Na3 V04 , and protease inhibitor mixture (Santa Cruz, Cat N. sc-29131) and sonicated with 2 × 10 s pulses. ..

Article Title: Lecithin:Cholesterol Acyltransferase Deficiency Protects against Cholesterol-induced Hepatic Endoplasmic Reticulum Stress in Mice *
Article Snippet: .. Liver samples (50–100 mg) were homogenized in radioimmune precipitation assay lysis buffer and protease inhibitor mixture (Santa Cruz Biotechnology, Santa Cruz, CA). .. Cytosolic and nuclear components of whole liver were isolated using the Compartment Protein Extraction kit (Chemicon catalog number 2145, Temecula, CA) as described previously ( ).

Article Title: Vinpocetine Attenuates Neointimal Hyperplasia in Diabetic Rat Carotid Arteries after Balloon Injury
Article Snippet: .. Western blot assay Cells were lysed in RIPA buffer with a protease inhibitor mixture (Santa Cruz), and protein concentrations were measured using the BCA protein assay kit (Beyotime, Shanghai, China). ..

Cell Culture:

Article Title: MicroRNA-mediated dysregulation of neural developmental genes in HPRT deficiency: clues for Lesch-Nyhan disease?
Article Snippet: .. Cells were cultured, lysed in six-well plates and the extracts were obtained using RIPA buffer with a protease inhibitor mixture (Santa Cruz, Inc.) (50 m m Tris–Cl, pH 8, 150 m m NaCl, 0.5% of deoxycholate, 0.1% SDS, 2 m m EDTA, 1% aprotinin, 1 m m PMSF, 1 m m sodium orthovanadate). ..

Purification:

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *
Article Snippet: .. For purification of mouse PECAM, a microsome fraction (∼50 mg of protein) was prepared by ultracentrifugation of mouse lung homogenates, solubilized in 1% Triton X-100 in TBS containing a protease inhibitor mixture, diluted with TBS until the final concentration of Triton X-100 was 0.3%, and subjected to immunoaffinity chromatography in which a de- N -glycosylated anti-PECAM antibody (200 μg; M-20; Santa Cruz Biotechnology) by peptide N -glycosidase F was coupled to 60 mg of tosylactivated Dynabeads M-280 (Invitrogen). .. After non-specifically bound proteins were eluted with a non-related peptide solution (100 μg/ml), the PECAM immunocomplex was digested with trypsin and chymotrypsin (Nacalai Tesque) in 50 m m ammonium bicarbonate at 37 °C for 16 h, boiled for 10 min, and treated with 30 μl of peptide N -glycosidase F at 37 °C for 16 h. N -Glycans released from 5 μg of pure PECAM were enriched with Oasis HLB Extraction Cartridges (Waters) and pyridylaminated as described previously ( , ).

Concentration Assay:

Article Title: Peptidomimetic blockade of MYB in acute myeloid leukemia
Article Snippet: .. Cells were lysed in RIPA buffer (Thermo Fisher) supplemented with a protease inhibitor mix comprised of AEBSF (0.5 mM concentration, Santa Cruz, SC-202041B), Bestatin (0.01 mM, Fisher/Alfa Aesar, J61106-MD), Leupeptin (0.1 mM, Santa Cruz, SC-295358B), and Pepstatin (0.001 mM, Santa Cruz, SC-45036A). .. Lysates were mechanically disrupted using Covaris S220 adaptive focused sonicator, according to the manufacturer’s instructions (Covaris, Woburn, CA).

Article Title: Interaction of Platelet Endothelial Cell Adhesion Molecule (PECAM) with α2,6-Sialylated Glycan Regulates Its Cell Surface Residency and Anti-apoptotic Role *
Article Snippet: .. For purification of mouse PECAM, a microsome fraction (∼50 mg of protein) was prepared by ultracentrifugation of mouse lung homogenates, solubilized in 1% Triton X-100 in TBS containing a protease inhibitor mixture, diluted with TBS until the final concentration of Triton X-100 was 0.3%, and subjected to immunoaffinity chromatography in which a de- N -glycosylated anti-PECAM antibody (200 μg; M-20; Santa Cruz Biotechnology) by peptide N -glycosidase F was coupled to 60 mg of tosylactivated Dynabeads M-280 (Invitrogen). .. After non-specifically bound proteins were eluted with a non-related peptide solution (100 μg/ml), the PECAM immunocomplex was digested with trypsin and chymotrypsin (Nacalai Tesque) in 50 m m ammonium bicarbonate at 37 °C for 16 h, boiled for 10 min, and treated with 30 μl of peptide N -glycosidase F at 37 °C for 16 h. N -Glycans released from 5 μg of pure PECAM were enriched with Oasis HLB Extraction Cartridges (Waters) and pyridylaminated as described previously ( , ).

BIA-KA:

Article Title: Vinpocetine Attenuates Neointimal Hyperplasia in Diabetic Rat Carotid Arteries after Balloon Injury
Article Snippet: .. Western blot assay Cells were lysed in RIPA buffer with a protease inhibitor mixture (Santa Cruz), and protein concentrations were measured using the BCA protein assay kit (Beyotime, Shanghai, China). ..

Western Blot:

Article Title: Vinpocetine Attenuates Neointimal Hyperplasia in Diabetic Rat Carotid Arteries after Balloon Injury
Article Snippet: .. Western blot assay Cells were lysed in RIPA buffer with a protease inhibitor mixture (Santa Cruz), and protein concentrations were measured using the BCA protein assay kit (Beyotime, Shanghai, China). ..

Lysis:

Article Title: Transforming Growth Factor-?1 (TGF-?1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts *
Article Snippet: .. Cells were grown to confluence in 35-mm dishes, and total cellular protein was extracted in radioimmune precipitation assay lysis buffer containing 1% protease inhibitor mixture, 1% PMSF, and 1% sodium orthovanadate (Santa Cruz Biotechnology, Inc.), as described previously ( ). .. Cell protein samples were incubated with anti-EGFR antibody-conjugated Magnabind beads (Thermo Scientific Pierce) overnight at 4 °C.

Article Title: Hyaluronan Orchestrates Transforming Growth Factor-β1-dependent Maintenance of Myofibroblast Phenotype
Article Snippet: .. Cells were then lysed using 1% protease inhibitor mixture, 1% phenymethylsulfonyl fluoride, and 1% sodium orthovanadate in RIPA lysis buffer (Santa Cruz Biotechnology, Inc.). ..

Article Title: Defective Sphingosine-1-phosphate metabolism is a druggable target in Huntington’s disease
Article Snippet: .. Total lysate preparation Tissues were homogenized in lysis buffer containing 20 mMTris, pH 7.4, 1% Nonidet P-40, 1 mM EDTA, 20 mMNaF, 2 mM Na3 V04 , and protease inhibitor mixture (Santa Cruz, Cat N. sc-29131) and sonicated with 2 × 10 s pulses. ..

Article Title: Lecithin:Cholesterol Acyltransferase Deficiency Protects against Cholesterol-induced Hepatic Endoplasmic Reticulum Stress in Mice *
Article Snippet: .. Liver samples (50–100 mg) were homogenized in radioimmune precipitation assay lysis buffer and protease inhibitor mixture (Santa Cruz Biotechnology, Santa Cruz, CA). .. Cytosolic and nuclear components of whole liver were isolated using the Compartment Protein Extraction kit (Chemicon catalog number 2145, Temecula, CA) as described previously ( ).

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  • 85
    Santa Cruz Biotechnology anti klk1b26 antibody
    Effect of miRNA preparations on reverse transcription (RT)-PCR for kallikrein 1b26 <t>(klk1b26)</t> mRNA . (A,B) Effect of miRNA preparation on the PCR product formation. RT-PCR was performed with the F21/R552 primer pair targeting the 5'-terminal region of klk1b26 mRNA (A) or with the F169/R552 primer pair targeting the middle region of the mRNA (B) in the presence of the indicated concentrations of miRNA prepared from either male or female submandibular glands (SMGs). Representative results are shown, and similar results were obtained in three independent experiments. (C) Effect of female miRNA preparation on the PCR product formation using various forward primers targeting the 5'-terminal region. PCR was carried out with each forward primer in combination with the R552 reverse primer in the absence (-) or presence (+) of miRNAs prepared from female SMGs (10 ng/μl). Sequences of the primers used are indicated in Table 1. Representative results are shown. Similar results were obtained in three independent experiments.
    Anti Klk1b26 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology l6 s1 drg
    HSV-GAD vector mediated expression of GAD 67 specific mRNA and protein in <t>L6-S1</t> DRGs. (A) GAD 67 mRNA/β-actin mRNA ratio in the <t>L6-S1</t> DRG determined by qRT-PCR was significantly increased in HSV-GAD-treated SCI rats compared with HSV-LacZ-treated
    L6 S1 Drg, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Santa Cruz Biotechnology rabbit klf5 antibody
    Mutation at each lysine residue preceding the DNA binding domain of <t>KLF5</t> does not prevent SMURF2 from degrading KLF5. A , alignment of all the lysine residues preceding the DNA binding domain of KLF5 from various species. Numbers indicate positions of
    Rabbit Klf5 Antibody, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Santa Cruz Biotechnology anti sall4 antibody chip grade
    A. Correlation between gene expression changes and DNA base composition observed with ZFC4-regulated genes at day 0 (top panel), day 2 (middle panel) and day 5 (bottom panel) of differentiation. ZFC4-regulated genes (see Figure 6E ) were divided into five equal categories according to their AT-content. Left panel: relative expression levels (log2 fold-change vs day 0 in WT cells) in WT and <t>Sall4</t> mutant cells. Right panel: Coefficient estimates (with 99% confidence intervals) describing the AT effect size. B. Correlation between gene expression changes and DNA base composition observed with ZFC1/2-regulated genes during differentiation, as described in panel A. C. PCA analysis of RNA-seq samples from WT and Sall4 mutant cell lines at day 0, 2 and 5 of differentiation. D. Scatter plot showing the relative expression levels of genes deregulated in differentiating ZFC4mut cells (see Figure 7B , red bars) correlating with their expression in S4KO cells at day 2 and 5 of differentiation.
    Anti Sall4 Antibody Chip Grade, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of miRNA preparations on reverse transcription (RT)-PCR for kallikrein 1b26 (klk1b26) mRNA . (A,B) Effect of miRNA preparation on the PCR product formation. RT-PCR was performed with the F21/R552 primer pair targeting the 5'-terminal region of klk1b26 mRNA (A) or with the F169/R552 primer pair targeting the middle region of the mRNA (B) in the presence of the indicated concentrations of miRNA prepared from either male or female submandibular glands (SMGs). Representative results are shown, and similar results were obtained in three independent experiments. (C) Effect of female miRNA preparation on the PCR product formation using various forward primers targeting the 5'-terminal region. PCR was carried out with each forward primer in combination with the R552 reverse primer in the absence (-) or presence (+) of miRNAs prepared from female SMGs (10 ng/μl). Sequences of the primers used are indicated in Table 1. Representative results are shown. Similar results were obtained in three independent experiments.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: Effect of miRNA preparations on reverse transcription (RT)-PCR for kallikrein 1b26 (klk1b26) mRNA . (A,B) Effect of miRNA preparation on the PCR product formation. RT-PCR was performed with the F21/R552 primer pair targeting the 5'-terminal region of klk1b26 mRNA (A) or with the F169/R552 primer pair targeting the middle region of the mRNA (B) in the presence of the indicated concentrations of miRNA prepared from either male or female submandibular glands (SMGs). Representative results are shown, and similar results were obtained in three independent experiments. (C) Effect of female miRNA preparation on the PCR product formation using various forward primers targeting the 5'-terminal region. PCR was carried out with each forward primer in combination with the R552 reverse primer in the absence (-) or presence (+) of miRNAs prepared from female SMGs (10 ng/μl). Sequences of the primers used are indicated in Table 1. Representative results are shown. Similar results were obtained in three independent experiments.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Masking of the interfering effect of female miRNA preparation on kallikrein 1b26 (klk1b26) translation by specific single-strand DNA, [15th-44th]ssDNA . (A) Masking of the activity in female miRNA preparation interfering klk1b26 translation by [15th-44th]ssDNA ([ 15 ]). The sequences of single-strand DNAs, [15th-44th]ssDNA and [169th-198th]ssDNA ([169]) (used as negative control), corresponded with the 15th to 44th and 169th to 198th nucleotide positions of klk1b26 mRNA, respectively, and are described in Table 1. Prior to the addition into the in vitro translation system, the female miRNA preparation was incubated with either of the single-strand DNAs. The in vitro translation reaction and the [ 35 S]klk1b26 protein analysis were carried out as described in Methods. Representative results are shown. Similar results were obtained from three independent experiments. (B) Quantitative determination of density of the [ 35 S]klk1b26 protein band. Values represent the mean ± SD (n = 3) of the relative density.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: Masking of the interfering effect of female miRNA preparation on kallikrein 1b26 (klk1b26) translation by specific single-strand DNA, [15th-44th]ssDNA . (A) Masking of the activity in female miRNA preparation interfering klk1b26 translation by [15th-44th]ssDNA ([ 15 ]). The sequences of single-strand DNAs, [15th-44th]ssDNA and [169th-198th]ssDNA ([169]) (used as negative control), corresponded with the 15th to 44th and 169th to 198th nucleotide positions of klk1b26 mRNA, respectively, and are described in Table 1. Prior to the addition into the in vitro translation system, the female miRNA preparation was incubated with either of the single-strand DNAs. The in vitro translation reaction and the [ 35 S]klk1b26 protein analysis were carried out as described in Methods. Representative results are shown. Similar results were obtained from three independent experiments. (B) Quantitative determination of density of the [ 35 S]klk1b26 protein band. Values represent the mean ± SD (n = 3) of the relative density.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Activity Assay, Negative Control, In Vitro, Incubation

    Inhibition of in vitro kallikrein 1b26 (klk1b26) translation by synthetic miRNAs partially complementary with the 5'-region of klk1b26 mRNA . (A) Sequences of miRNAs and their complementary sites in the 5'-region of klk1b26 mRNA. The numbers above the klk1b26 mRNA sequence indicate the nucleotide positions from the transcription start site (GenBank: NM 010644 ). (B) Inhibition of in vitro translation of klk1b26 by the miRNAs and antisense RNA of F21 forward primer (asR-F21). Messenger RNA (500 ng) purified from male submandibular glands (SMGs) was preincubated with or without miR-325, miR-1497a or asR-F21 (10 μM each) in a translation mixture. The mixture was then incubated with a reticulocyte lysate for in vitro protein synthesis, and [ 35 S]methionine-labeled klk1b26 protein was analyzed by SDS-PAGE as described in Methods. N, 325, 1497a, and asR stand for the absence of synthetic RNA, miR-325, miR-1497a, and asR-F21, respectively. Representative results are shown. Similar results were obtained in three independent experiments. (C) Effects of synthetic miRNAs and antisense RNA of F21 forward primer (asR-F21) on reverse transcription (RT)-PCR for klk1b26 mRNA using various forward primers targeting the 5'-terminal region. Sequences of the primers are described in Table 1. PCR was carried out with each forward primer in combination with the R552 reverse primer in the absence or presence of the synthetic RNAs. N, 325, 1497a, and asR indicated the absence of synthetic RNA (3 μM), miR-325, miR-1497a, and asR-F21, respectively. Representative results are shown. Similar results were obtained in three independent experiments.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: Inhibition of in vitro kallikrein 1b26 (klk1b26) translation by synthetic miRNAs partially complementary with the 5'-region of klk1b26 mRNA . (A) Sequences of miRNAs and their complementary sites in the 5'-region of klk1b26 mRNA. The numbers above the klk1b26 mRNA sequence indicate the nucleotide positions from the transcription start site (GenBank: NM 010644 ). (B) Inhibition of in vitro translation of klk1b26 by the miRNAs and antisense RNA of F21 forward primer (asR-F21). Messenger RNA (500 ng) purified from male submandibular glands (SMGs) was preincubated with or without miR-325, miR-1497a or asR-F21 (10 μM each) in a translation mixture. The mixture was then incubated with a reticulocyte lysate for in vitro protein synthesis, and [ 35 S]methionine-labeled klk1b26 protein was analyzed by SDS-PAGE as described in Methods. N, 325, 1497a, and asR stand for the absence of synthetic RNA, miR-325, miR-1497a, and asR-F21, respectively. Representative results are shown. Similar results were obtained in three independent experiments. (C) Effects of synthetic miRNAs and antisense RNA of F21 forward primer (asR-F21) on reverse transcription (RT)-PCR for klk1b26 mRNA using various forward primers targeting the 5'-terminal region. Sequences of the primers are described in Table 1. PCR was carried out with each forward primer in combination with the R552 reverse primer in the absence or presence of the synthetic RNAs. N, 325, 1497a, and asR indicated the absence of synthetic RNA (3 μM), miR-325, miR-1497a, and asR-F21, respectively. Representative results are shown. Similar results were obtained in three independent experiments.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Inhibition, In Vitro, Sequencing, Purification, Incubation, Labeling, SDS Page, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Reverse transcription (RT)-PCR for kallikrein 1b26 (klk1b26) mRNA by using various forward primers targeting the 5'-terminal region and middle region of the mRNA . (A) Positions of forward primers indicated the ordinal numbers of the nucleotides in the klk1b26 mRNA sequence (NM 010644) from its 5'-terminal end. Each forward primer was used in combination with the R552 reverse primer. Sequences of those primers are described in Table 1. (B, C) Total RNAs of ICR mouse submandibular glands (SMGs) were prepared from the following sources: male, castrated male (Cast), Cast + 5α-dihydrotestosterone (DHT), female, and female + DHT. (B) RT-PCR with primer pairs targeting the 5'-terminal region of klk1b26 mRNA was carried out by using either the primer pairs of F21 forward primer and R552 reverse primer or F21 forward primer and R609 reverse primer. (C) RT-PCR was carried out by using the F169 forward primer and R552 reverse primer or the F169 forward primer and R609 reverse primer targeting the middle region of klk1b26 mRNA. In (A-C), representative results are shown; similar results were obtained in three independent experiments.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: Reverse transcription (RT)-PCR for kallikrein 1b26 (klk1b26) mRNA by using various forward primers targeting the 5'-terminal region and middle region of the mRNA . (A) Positions of forward primers indicated the ordinal numbers of the nucleotides in the klk1b26 mRNA sequence (NM 010644) from its 5'-terminal end. Each forward primer was used in combination with the R552 reverse primer. Sequences of those primers are described in Table 1. (B, C) Total RNAs of ICR mouse submandibular glands (SMGs) were prepared from the following sources: male, castrated male (Cast), Cast + 5α-dihydrotestosterone (DHT), female, and female + DHT. (B) RT-PCR with primer pairs targeting the 5'-terminal region of klk1b26 mRNA was carried out by using either the primer pairs of F21 forward primer and R552 reverse primer or F21 forward primer and R609 reverse primer. (C) RT-PCR was carried out by using the F169 forward primer and R552 reverse primer or the F169 forward primer and R609 reverse primer targeting the middle region of klk1b26 mRNA. In (A-C), representative results are shown; similar results were obtained in three independent experiments.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

    5' Rapid amplification of cDNA ends (RACE) analysis of kallikrein 1b26 (klk1b26) mRNAs from male and female submandibular glands (SMGs) and effect of incubation with SMG extracts on the mRNA . (A) Antisense chains of 5' RACE products were prepared and analyzed as described in Methods. Each signal is indicated by color: green = A; red, = T; black = G; blue = C. (B) Degradation of klk1b26 mRNA by incubation with the SMG extracts. Total RNA from male SMGs (21.5 μg) was incubated at 37°C for 30 min with an extract prepared from male or female mouse SMGs as described in Methods. After the incubation the total RNA was again purified with the Micro-to-Midi Total RNA Purification System (Invitrogen) and was eluted to 20 μl, quantitatively. First-strand DNAs were prepared with 280 ng of the total RNAs thus treated with SMG extracts and reverse transcription (RT)-PCR was carried out by using primer pairs F21/R552 or F169/R552. Representative results are shown. Similar results were obtained in three independent experiments. Quantitative determination of density of the PCR products was made by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/ ). Relative densities of the PCR signals with total RNAs treated by 8 μg/μl each of male SMG extract and female SMG extract were estimated to be 7.36 ± 0.40 and 7.44 ± 0.51, respectively (mean ± SD; n = 3 each). There was no significant difference in the degrading activities for the mRNA between the male and female SMG extracts.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: 5' Rapid amplification of cDNA ends (RACE) analysis of kallikrein 1b26 (klk1b26) mRNAs from male and female submandibular glands (SMGs) and effect of incubation with SMG extracts on the mRNA . (A) Antisense chains of 5' RACE products were prepared and analyzed as described in Methods. Each signal is indicated by color: green = A; red, = T; black = G; blue = C. (B) Degradation of klk1b26 mRNA by incubation with the SMG extracts. Total RNA from male SMGs (21.5 μg) was incubated at 37°C for 30 min with an extract prepared from male or female mouse SMGs as described in Methods. After the incubation the total RNA was again purified with the Micro-to-Midi Total RNA Purification System (Invitrogen) and was eluted to 20 μl, quantitatively. First-strand DNAs were prepared with 280 ng of the total RNAs thus treated with SMG extracts and reverse transcription (RT)-PCR was carried out by using primer pairs F21/R552 or F169/R552. Representative results are shown. Similar results were obtained in three independent experiments. Quantitative determination of density of the PCR products was made by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/ ). Relative densities of the PCR signals with total RNAs treated by 8 μg/μl each of male SMG extract and female SMG extract were estimated to be 7.36 ± 0.40 and 7.44 ± 0.51, respectively (mean ± SD; n = 3 each). There was no significant difference in the degrading activities for the mRNA between the male and female SMG extracts.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Rapid Amplification of cDNA Ends, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Effects of exo-mRNA fraction on reverse transcription (RT)-PCR for kallikrein 1b26 (klk1b26) mRNA and inhibition of klk1b26 translation by miRNA preparation from female submandibular glands (SMGs) . (A) Effect of exo-mRNA fraction on RT-PCR with a primer pair targeting the 5'-terminal region of klk1b26 mRNA. RT-PCR was carried out with the primer pair F21 forward and R552 reverse in the presence or absence of the exo-mRNA fraction (100 ng/μl) from male or female SMGs. (B) Quantitative determination of density of klk1b26 PCR products in Figure 4A was measured by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/ ). Values are averages of the duplicate assay. (C, D) Inhibition of klk1b26 translation in vitro by miRNA preparation from female mouse SMGs. After the preincubation of mRNAs purified from male SMGs with miRNA preparation from male or female SMGs, the in vitro translation was performed and klk1b26 protein synthesized was analyzed as described in Methods. Representative results are shown. Similar results were obtained from three independent experiments (C). Quantitative determination of density of the [ 35 S]klk1b26 protein band. Values represent the mean ± SD (n = 3) of the relative density (D). (E) Representative autoradiograms of [ 35 S]methionine-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein on the SDS-PAGE gels. In vitro translation was performed as described in Methods. GAPDH protein was immunoprecipitated with anti-GAPDH antibody. (F) Quantitative determination of density of the [ 35 S]klk1b26 protein band. Values are the mean ± SD (n = 3) of the relative density.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: Effects of exo-mRNA fraction on reverse transcription (RT)-PCR for kallikrein 1b26 (klk1b26) mRNA and inhibition of klk1b26 translation by miRNA preparation from female submandibular glands (SMGs) . (A) Effect of exo-mRNA fraction on RT-PCR with a primer pair targeting the 5'-terminal region of klk1b26 mRNA. RT-PCR was carried out with the primer pair F21 forward and R552 reverse in the presence or absence of the exo-mRNA fraction (100 ng/μl) from male or female SMGs. (B) Quantitative determination of density of klk1b26 PCR products in Figure 4A was measured by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/ ). Values are averages of the duplicate assay. (C, D) Inhibition of klk1b26 translation in vitro by miRNA preparation from female mouse SMGs. After the preincubation of mRNAs purified from male SMGs with miRNA preparation from male or female SMGs, the in vitro translation was performed and klk1b26 protein synthesized was analyzed as described in Methods. Representative results are shown. Similar results were obtained from three independent experiments (C). Quantitative determination of density of the [ 35 S]klk1b26 protein band. Values represent the mean ± SD (n = 3) of the relative density (D). (E) Representative autoradiograms of [ 35 S]methionine-labeled glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein on the SDS-PAGE gels. In vitro translation was performed as described in Methods. GAPDH protein was immunoprecipitated with anti-GAPDH antibody. (F) Quantitative determination of density of the [ 35 S]klk1b26 protein band. Values are the mean ± SD (n = 3) of the relative density.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Inhibition, Polymerase Chain Reaction, In Vitro, Purification, Synthesized, Labeling, SDS Page, Immunoprecipitation

    Effects of castration and 5α-dihydrotestosterone (DHT) administration on the activity in the submandibular gland (SMG) miRNA preparation interfering kallikrein 1b26 (klk1b26) translation . (A) MicroRNAs were prepared as described in Methods from SMGs of castrated mouse, DHT-administered castrated mouse and DHT-administered female mouse, respectively. One miRNA sample was prepared from SMGs from one mouse. The effects of these miRNA preparations (90 ng/μl each) on klk1b26 translation were analyzed as described in the legend for Figure 4C. Representative results are shown. Similar results were obtained in three independent experiments. (B) Quantitative determination of density of the [ 35 S]klk1b26 protein band by computer-assisted image analysis of the autoradiograms. Values represent the mean ± SD (n = 3 animals) of the relative density.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: Effects of castration and 5α-dihydrotestosterone (DHT) administration on the activity in the submandibular gland (SMG) miRNA preparation interfering kallikrein 1b26 (klk1b26) translation . (A) MicroRNAs were prepared as described in Methods from SMGs of castrated mouse, DHT-administered castrated mouse and DHT-administered female mouse, respectively. One miRNA sample was prepared from SMGs from one mouse. The effects of these miRNA preparations (90 ng/μl each) on klk1b26 translation were analyzed as described in the legend for Figure 4C. Representative results are shown. Similar results were obtained in three independent experiments. (B) Quantitative determination of density of the [ 35 S]klk1b26 protein band by computer-assisted image analysis of the autoradiograms. Values represent the mean ± SD (n = 3 animals) of the relative density.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Activity Assay

    Kallikrein 1b26 (klk1b26) protein and its mRNA levels in submandibular glands (SMGs) of male and female mice . (A) Western blot analysis of klk1b26 proteins in SMGs. One sample of SMG protein extract was prepared from one mouse (two SMGs). The protein extracts from male and female SMGs (10 ng protein each) were subjected to SDS-PAGE (16% non-reducing gel), and blotted onto a polyvinylidene difluoride (PVDF) filter. The klk1b26 protein was detected with anti-klk1b26 polyclonal antibody. Quantitative determination of the klk1b26 signals was made by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/ ). The klk1b26 protein ratio of male SMG to female SMG was estimated to be 9.16 ± 1.64 (mean ± SD; n = 3 mice). (B) Quantitative reverse transcription (RT)-PCR analysis of klk1b26 mRNA in SMGs. First-strand DNAs were prepared with 200 ng of total RNAs from male and female SMGs by using oligo(dT) primer. PCR was performed with primer pairs (F169/R552) targeting the middle region (169th to 552nd) of klk1b26 mRNA. The PCR conditions used were as follows: 94°C for 30 s, 55°C for 30 s, and 68°C for 120 s for denaturation, annealing, and elongation, respectively. (C) Quantitative determination of density of klk1b26 PCR products was made by computer-assisted image analysis, NIH image. The klk1b26 mRNA level of male SMG was estimated to be sevenfold higher than that of female SMG from the results of duplicate experiments.

    Journal: Biology of Sex Differences

    Article Title: Interference of kallikrein 1b26 (klk1b26) translation by microRNA specifically expressed in female mouse submandibular glands: an additional mechanism for sexual dimorphism of klk1b26 protein in the glands

    doi: 10.1186/2042-6410-2-13

    Figure Lengend Snippet: Kallikrein 1b26 (klk1b26) protein and its mRNA levels in submandibular glands (SMGs) of male and female mice . (A) Western blot analysis of klk1b26 proteins in SMGs. One sample of SMG protein extract was prepared from one mouse (two SMGs). The protein extracts from male and female SMGs (10 ng protein each) were subjected to SDS-PAGE (16% non-reducing gel), and blotted onto a polyvinylidene difluoride (PVDF) filter. The klk1b26 protein was detected with anti-klk1b26 polyclonal antibody. Quantitative determination of the klk1b26 signals was made by computer-assisted image analysis (NIH image; http://rsbweb.nih.gov/nih-image/ ). The klk1b26 protein ratio of male SMG to female SMG was estimated to be 9.16 ± 1.64 (mean ± SD; n = 3 mice). (B) Quantitative reverse transcription (RT)-PCR analysis of klk1b26 mRNA in SMGs. First-strand DNAs were prepared with 200 ng of total RNAs from male and female SMGs by using oligo(dT) primer. PCR was performed with primer pairs (F169/R552) targeting the middle region (169th to 552nd) of klk1b26 mRNA. The PCR conditions used were as follows: 94°C for 30 s, 55°C for 30 s, and 68°C for 120 s for denaturation, annealing, and elongation, respectively. (C) Quantitative determination of density of klk1b26 PCR products was made by computer-assisted image analysis, NIH image. The klk1b26 mRNA level of male SMG was estimated to be sevenfold higher than that of female SMG from the results of duplicate experiments.

    Article Snippet: Proteins synthesized in the in vitro system were mixed with anti-klk1b26 antibody or anti-GAPDH antibody (sc-25778; Santa Cruz Biotechnology, Santa Cruz, CA, USA) preconjugated to protein G-beads (no. 22851; Pierce Biotechnology, Rockford, IL, USA) and incubated for 2 h in PBS/1% ovalbumin containing 1/100 × protease inhibitor cocktail (no. 535140, Calbiochem).

    Techniques: Mouse Assay, Western Blot, SDS Page, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    HSV-GAD vector mediated expression of GAD 67 specific mRNA and protein in L6-S1 DRGs. (A) GAD 67 mRNA/β-actin mRNA ratio in the L6-S1 DRG determined by qRT-PCR was significantly increased in HSV-GAD-treated SCI rats compared with HSV-LacZ-treated

    Journal: Gene therapy

    Article Title: Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord injured rats

    doi: 10.1038/gt.2009.5

    Figure Lengend Snippet: HSV-GAD vector mediated expression of GAD 67 specific mRNA and protein in L6-S1 DRGs. (A) GAD 67 mRNA/β-actin mRNA ratio in the L6-S1 DRG determined by qRT-PCR was significantly increased in HSV-GAD-treated SCI rats compared with HSV-LacZ-treated

    Article Snippet: L6-S1 DRG were sonicated in RIPA buffer containing 1X RIPA Lysis buffer, PMSF, sodium orthovanadate, and protease inhibitor cocktail (0.3 ml/100 mg tissue, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Plasmid Preparation, Expressing, Quantitative RT-PCR

    HSV vector-mediated transgene expression in rat L6-S1 DRGs after administration of vector to the bladder wall. (A) GFP expression in L6-S1 DRG in a HSV-GAD-treated SCI rat. A significant number of GFP-positive cells were seen in L6-S1 DRG 3 weeks after

    Journal: Gene therapy

    Article Title: Herpes simplex virus vector-mediated gene delivery of glutamic acid decarboxylase reduces detrusor overactivity in spinal cord injured rats

    doi: 10.1038/gt.2009.5

    Figure Lengend Snippet: HSV vector-mediated transgene expression in rat L6-S1 DRGs after administration of vector to the bladder wall. (A) GFP expression in L6-S1 DRG in a HSV-GAD-treated SCI rat. A significant number of GFP-positive cells were seen in L6-S1 DRG 3 weeks after

    Article Snippet: L6-S1 DRG were sonicated in RIPA buffer containing 1X RIPA Lysis buffer, PMSF, sodium orthovanadate, and protease inhibitor cocktail (0.3 ml/100 mg tissue, Santa Cruz Biotechnology, Santa Cruz, CA).

    Techniques: Plasmid Preparation, Expressing

    Mutation at each lysine residue preceding the DNA binding domain of KLF5 does not prevent SMURF2 from degrading KLF5. A , alignment of all the lysine residues preceding the DNA binding domain of KLF5 from various species. Numbers indicate positions of

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: Mutation at each lysine residue preceding the DNA binding domain of KLF5 does not prevent SMURF2 from degrading KLF5. A , alignment of all the lysine residues preceding the DNA binding domain of KLF5 from various species. Numbers indicate positions of

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques: Mutagenesis, Binding Assay

    SMURF2 degrades and destabilizes KLF5. A , reduced level of HA-KLF5 upon Myc-SMURF2 cotransfection. HEK293T cells were cotransfected with HA-KLF5 and either vector alone or Myc-SMURF2. Whole cell lysates were immunoblotted with mouse HA, Myc, or β-actin

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: SMURF2 degrades and destabilizes KLF5. A , reduced level of HA-KLF5 upon Myc-SMURF2 cotransfection. HEK293T cells were cotransfected with HA-KLF5 and either vector alone or Myc-SMURF2. Whole cell lysates were immunoblotted with mouse HA, Myc, or β-actin

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques: Cotransfection, Plasmid Preparation

    SMURF2 ubiquitinates KLF5. A , ubiquitination of overexpressed KLF5 with HA-tagged ubiquitin. HEK293T cells were cotransfected with HA-ubiquitin ( HA-Ubi ) and either Myc-SMURF2 ( S ), KLF5 ( K ), or both ( S + K ) and treated with MG132. The corresponding lysates

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: SMURF2 ubiquitinates KLF5. A , ubiquitination of overexpressed KLF5 with HA-tagged ubiquitin. HEK293T cells were cotransfected with HA-ubiquitin ( HA-Ubi ) and either Myc-SMURF2 ( S ), KLF5 ( K ), or both ( S + K ) and treated with MG132. The corresponding lysates

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques:

    KLF5 colocalizes with SMURF2. COS-1 cells were transfected with HA-KLF5 and Myc-SMURF2, treated with MG132 to stabilize HA-KLF5, and stained with chicken anti-HA and rabbit anti-Myc, followed by FITC-conjugated donkey α-chicken and Cy5-conjugated

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: KLF5 colocalizes with SMURF2. COS-1 cells were transfected with HA-KLF5 and Myc-SMURF2, treated with MG132 to stabilize HA-KLF5, and stained with chicken anti-HA and rabbit anti-Myc, followed by FITC-conjugated donkey α-chicken and Cy5-conjugated

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques: Transfection, Staining

    KLF5 interacts with SMURF2. A , SMURF2 binds KLF5 in a yeast two-hybrid assay through the carboxyl terminus of KLF5. The indicated SMURF2 or full-length or truncated KLF5 constructs (or the corresponding vector alone) were transformed in yeast and selected

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: KLF5 interacts with SMURF2. A , SMURF2 binds KLF5 in a yeast two-hybrid assay through the carboxyl terminus of KLF5. The indicated SMURF2 or full-length or truncated KLF5 constructs (or the corresponding vector alone) were transformed in yeast and selected

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques: Y2H Assay, Construct, Plasmid Preparation, Transformation Assay

    SMURF2 inhibits the transcriptional and pro-proliferative activities of KLF5. A , depletion of SMURF2 increases the expression of KLF5 target genes. COS-1 cells were transfected with either control siRNA or the Trilencer SMURF2 siRNA mixture from Origene

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: SMURF2 inhibits the transcriptional and pro-proliferative activities of KLF5. A , depletion of SMURF2 increases the expression of KLF5 target genes. COS-1 cells were transfected with either control siRNA or the Trilencer SMURF2 siRNA mixture from Origene

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques: Expressing, Transfection

    Mutation at single lysine residues within KLF5's DNA binding domain does not prevent SMURF2 from degrading KLF5. A , comparison of the zinc finger DNA-binding domains of mouse KLF5 and KLF4. The numbers indicate the positions of lysine residues. The two

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: Mutation at single lysine residues within KLF5's DNA binding domain does not prevent SMURF2 from degrading KLF5. A , comparison of the zinc finger DNA-binding domains of mouse KLF5 and KLF4. The numbers indicate the positions of lysine residues. The two

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques: Mutagenesis, Binding Assay

    Endogenous KLF5 colocalizes with SMURF2. DLD-1 cells were treated with MG132 to stabilize cellular proteins and stained with rabbit anti-SMURF2 and mouse anti-KLF5, followed by RRX-conjugated donkey α-rabbit and FITC-conjugated donkey α-mouse

    Journal: The Journal of Biological Chemistry

    Article Title: The E3 Ubiquitin Ligase SMAD Ubiquitination Regulatory Factor 2 Negatively Regulates Kr?ppel-like Factor 5 Protein *

    doi: 10.1074/jbc.M111.258707

    Figure Lengend Snippet: Endogenous KLF5 colocalizes with SMURF2. DLD-1 cells were treated with MG132 to stabilize cellular proteins and stained with rabbit anti-SMURF2 and mouse anti-KLF5, followed by RRX-conjugated donkey α-rabbit and FITC-conjugated donkey α-mouse

    Article Snippet: The lysates were denatured by boiling for 10 min, diluted in five volumes of dilution buffer (10 m m Tris-HCl (pH7.4), 150 m m NaCl, 1% Triton X-100, 2 m m EDTA, complete protease inhibitor mixture), and immunoprecipitated with the mixture of a rabbit KLF5 antibody ( ) and commercial rabbit KLF5 antibody from Santa Cruz (SC22797), followed by incubation with protein A beads (Upstate).

    Techniques: Staining

    A. Correlation between gene expression changes and DNA base composition observed with ZFC4-regulated genes at day 0 (top panel), day 2 (middle panel) and day 5 (bottom panel) of differentiation. ZFC4-regulated genes (see Figure 6E ) were divided into five equal categories according to their AT-content. Left panel: relative expression levels (log2 fold-change vs day 0 in WT cells) in WT and Sall4 mutant cells. Right panel: Coefficient estimates (with 99% confidence intervals) describing the AT effect size. B. Correlation between gene expression changes and DNA base composition observed with ZFC1/2-regulated genes during differentiation, as described in panel A. C. PCA analysis of RNA-seq samples from WT and Sall4 mutant cell lines at day 0, 2 and 5 of differentiation. D. Scatter plot showing the relative expression levels of genes deregulated in differentiating ZFC4mut cells (see Figure 7B , red bars) correlating with their expression in S4KO cells at day 2 and 5 of differentiation.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: A. Correlation between gene expression changes and DNA base composition observed with ZFC4-regulated genes at day 0 (top panel), day 2 (middle panel) and day 5 (bottom panel) of differentiation. ZFC4-regulated genes (see Figure 6E ) were divided into five equal categories according to their AT-content. Left panel: relative expression levels (log2 fold-change vs day 0 in WT cells) in WT and Sall4 mutant cells. Right panel: Coefficient estimates (with 99% confidence intervals) describing the AT effect size. B. Correlation between gene expression changes and DNA base composition observed with ZFC1/2-regulated genes during differentiation, as described in panel A. C. PCA analysis of RNA-seq samples from WT and Sall4 mutant cell lines at day 0, 2 and 5 of differentiation. D. Scatter plot showing the relative expression levels of genes deregulated in differentiating ZFC4mut cells (see Figure 7B , red bars) correlating with their expression in S4KO cells at day 2 and 5 of differentiation.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Expressing, Mutagenesis, RNA Sequencing Assay

    SALL4-mediated transcriptional regulation in relation to DNA base composition A. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between S4KO, ZFC4mut and ZFC4 Δ ESCs. ZFC4-regulated genes are indicated in red, and ZFC4-independent genes in grey. B. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-regulated genes divided into five equal categories according to AT-content. TSS: Transcription start site, TES: Transcription end site. C, D. Correlation between gene mis-regulation (log2 fold-change vs WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (C) and ZFC4-independent (D) genes were divided into five equal categories depending on their AT-content. E. Diagram representing Sall4 knockout ESC lines carrying SALL4 or EGFP (control) expression constructs under control of a doxycycline-inducible promoter. F. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq following a 48h doxycycline induction in the ESC lines presented in panel E. SALL4-responsive genes are indicated in blue, and EGFP-responsive genes in green. G, H. Correlation between SALL4-induced gene expression changes and DNA base composition. SALL4-responsive (G) and ZFC4-regulated (H) genes were divided into five equal categories depending on their AT-content, and their relative expression levels were analysed in the indicated ESC lines. I. Diagram showing the protocol used to characterise early differentiation of WT ESCs. J. Venn diagram showing the overlap between genes changing during early differentiation of WT cells (day 0 vs day 2) with genes de-regulated in Sall4 mutant ESCs. Genes were divided into three categories: SALL4-independent genes (light blue), SALL4-dependent genes controlled by ZFC4 (red) and SALL4-dependent genes not controlled by ZFC4 (grey). K. Correlation between gene expression changes occurring during early differentiation and DNA base composition in WT cells. SALL4-independent genes (light blue), SALL4-dependent genes controlled by ZFC4 (red) and SALL4-dependent genes not controlled by ZFC4 (grey) were divided into five equal categories depending on their AT-content, and their relative expression levels were analysed at day 2 of differentiation.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: SALL4-mediated transcriptional regulation in relation to DNA base composition A. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between S4KO, ZFC4mut and ZFC4 Δ ESCs. ZFC4-regulated genes are indicated in red, and ZFC4-independent genes in grey. B. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-regulated genes divided into five equal categories according to AT-content. TSS: Transcription start site, TES: Transcription end site. C, D. Correlation between gene mis-regulation (log2 fold-change vs WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (C) and ZFC4-independent (D) genes were divided into five equal categories depending on their AT-content. E. Diagram representing Sall4 knockout ESC lines carrying SALL4 or EGFP (control) expression constructs under control of a doxycycline-inducible promoter. F. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq following a 48h doxycycline induction in the ESC lines presented in panel E. SALL4-responsive genes are indicated in blue, and EGFP-responsive genes in green. G, H. Correlation between SALL4-induced gene expression changes and DNA base composition. SALL4-responsive (G) and ZFC4-regulated (H) genes were divided into five equal categories depending on their AT-content, and their relative expression levels were analysed in the indicated ESC lines. I. Diagram showing the protocol used to characterise early differentiation of WT ESCs. J. Venn diagram showing the overlap between genes changing during early differentiation of WT cells (day 0 vs day 2) with genes de-regulated in Sall4 mutant ESCs. Genes were divided into three categories: SALL4-independent genes (light blue), SALL4-dependent genes controlled by ZFC4 (red) and SALL4-dependent genes not controlled by ZFC4 (grey). K. Correlation between gene expression changes occurring during early differentiation and DNA base composition in WT cells. SALL4-independent genes (light blue), SALL4-dependent genes controlled by ZFC4 (red) and SALL4-dependent genes not controlled by ZFC4 (grey) were divided into five equal categories depending on their AT-content, and their relative expression levels were analysed at day 2 of differentiation.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: RNA Sequencing Assay, Mutagenesis, Knock-Out, Expressing, Construct

    Characterisation of SALL4 C2H2 zinc-finger clusters 1 and 2 in ESCs A. Diagram showing the in frame deletion of SALL4 within the Sall4 coding sequence, generated by CRISPR/Cas9. B. SALL4 ZFC1-2Δ localisation determined by immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars: 3μm. C. Heatmap and profile plot showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. D. Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit +/- 250bp) in WT (blue) and ZFC1-2 Δ (purple) ESCs. E. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between ZFC4mut and ZFC1-2 Δ ESCs. ZFC4-regulated genes are indicated in red and ZFC1/2-regulated genes in purple. F. Correlation between gene mis-regulation (log2 fold-change vs WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes were divided into five equal categories depending on their AT-content. G. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: Characterisation of SALL4 C2H2 zinc-finger clusters 1 and 2 in ESCs A. Diagram showing the in frame deletion of SALL4 within the Sall4 coding sequence, generated by CRISPR/Cas9. B. SALL4 ZFC1-2Δ localisation determined by immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars: 3μm. C. Heatmap and profile plot showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. D. Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit +/- 250bp) in WT (blue) and ZFC1-2 Δ (purple) ESCs. E. Venn diagram showing the overlap of differentially expressed genes detected by RNA-seq between ZFC4mut and ZFC1-2 Δ ESCs. ZFC4-regulated genes are indicated in red and ZFC1/2-regulated genes in purple. F. Correlation between gene mis-regulation (log2 fold-change vs WT ) and DNA base composition in Sall4 mutant ESCs. ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes were divided into five equal categories depending on their AT-content. G. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Sequencing, Generated, CRISPR, Immunofluorescence, Staining, Chromatin Immunoprecipitation, RNA Sequencing Assay, Mutagenesis, Cell Culture

    Characterisation of SALL4 C2H2 zinc-finger clusters during neuronal differentiation A. Diagram of the RNA-seq timecourse experiment comparing the differentiation potential of WT and Sall4 mutant ESCs. B. Differential gene expression analysis between WT and Sall4 mutant cell lines during neuronal differentiation (day 2 and 5). Additional WT replicates were used a s a control (WT vs WT ). C. Scatter plot showing the relative expression levels of genes deregulated in differentiating S4KO cells (see Figure 7B , grey bars) correlating with their expression in ZFC4mut cells at day 2 and 5 of differentiation. D. Relative expression levels (log2 fold-change vs WT ) of genes associated with the GO term “positive regulation of neuron differentiation” (GO:0045666) in Sall4 mutant cell lines at day 2 and 5 of differentiation. Additional WT replicates were used as a control (WT vs WT).

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: Characterisation of SALL4 C2H2 zinc-finger clusters during neuronal differentiation A. Diagram of the RNA-seq timecourse experiment comparing the differentiation potential of WT and Sall4 mutant ESCs. B. Differential gene expression analysis between WT and Sall4 mutant cell lines during neuronal differentiation (day 2 and 5). Additional WT replicates were used a s a control (WT vs WT ). C. Scatter plot showing the relative expression levels of genes deregulated in differentiating S4KO cells (see Figure 7B , grey bars) correlating with their expression in ZFC4mut cells at day 2 and 5 of differentiation. D. Relative expression levels (log2 fold-change vs WT ) of genes associated with the GO term “positive regulation of neuron differentiation” (GO:0045666) in Sall4 mutant cell lines at day 2 and 5 of differentiation. Additional WT replicates were used as a control (WT vs WT).

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: RNA Sequencing Assay, Mutagenesis, Expressing

    Identification of novel AT-binding proteins in embryonic stem cells by DNA pulldownmass spectrometry A. Results of a DNA pulldown-mass spectrometry screen with SILAC-labelled ESC nuclear protein extracts, comparing AT-rich DNA probes (AT-1, AT-2) with control probes having interrupted AT-runs (Ctrl-1, Ctrl-2). B. DNA pulldown with AT-rich (AT-2) or control (Ctrl-2) probes followed by Western blot analysis for SALL4 and NuRD components using wild-type (WT ) or Sall4 knockout ( S4KO ) ESC protein extracts. C. Protein alignment of mouse SALL family members indicating conserved protein domains, including C2H2 zinc-finger clusters (ZFC1-4). D. Diagram showing the mutations or deletion introduced within SALL4 ZFC4 by CRISPR/Cas9. E. DNA pulldown with AT-rich (AT-3) or control (Ctrl-3) probe followed by Western blot analysis for SALL4 using WT or Sall4 ZFC4mut/ Δ ESC protein extracts. SALL4 levels were quantified and normalised to input. Data points indicate independent replicate experiments, and error bars standard deviation. F. SALL4 immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars: 3μm.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: Identification of novel AT-binding proteins in embryonic stem cells by DNA pulldownmass spectrometry A. Results of a DNA pulldown-mass spectrometry screen with SILAC-labelled ESC nuclear protein extracts, comparing AT-rich DNA probes (AT-1, AT-2) with control probes having interrupted AT-runs (Ctrl-1, Ctrl-2). B. DNA pulldown with AT-rich (AT-2) or control (Ctrl-2) probes followed by Western blot analysis for SALL4 and NuRD components using wild-type (WT ) or Sall4 knockout ( S4KO ) ESC protein extracts. C. Protein alignment of mouse SALL family members indicating conserved protein domains, including C2H2 zinc-finger clusters (ZFC1-4). D. Diagram showing the mutations or deletion introduced within SALL4 ZFC4 by CRISPR/Cas9. E. DNA pulldown with AT-rich (AT-3) or control (Ctrl-3) probe followed by Western blot analysis for SALL4 using WT or Sall4 ZFC4mut/ Δ ESC protein extracts. SALL4 levels were quantified and normalised to input. Data points indicate independent replicate experiments, and error bars standard deviation. F. SALL4 immunofluorescence in the indicated ESC lines. DNA was stained with DAPI, showing dense clusters of AT-rich pericentric chromatin. Scale bars: 3μm.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Binding Assay, Mass Spectrometry, Western Blot, Knock-Out, CRISPR, Standard Deviation, Immunofluorescence, Staining

    A, B. Venn diagrams showing the overlap between proteins identified by DNA pulldown-mass spectrometry in independent replicate experiments (A), or using unrelated AT-rich DNA probes (B). C. AT-rich DNA probe containing a single AT-run (AT-3) and associated control probe (Ctrl-3). D. DNA pulldown with AT-rich (AT-1, AT-2, AT-3) or control (Ctrl-1, Ctrl-2, Ctrl-3) probes followed by Western blot analysis for SALL4 using WT ESC protein extracts. E. Protein alignment and consensus sequence of C2H2 zinc-finger cluster 4 (ZFC4) in the mouse SALL protein family. ZFC4 is absent in SALL2. F. Western blot quantification of SALL4 expression levels in S4KO and ZFC4mut/ Δ ESCs, normalised to HDAC1 expression and relative to WT ESC levels. Data points indicate independent replicate experiments and error bars standard deviation. G. SALL4 co-immunoprecipitation with SALL1 and NuRD components in WT, S4KO (negative control) and ZFC4mut/ Δ ESCs.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: A, B. Venn diagrams showing the overlap between proteins identified by DNA pulldown-mass spectrometry in independent replicate experiments (A), or using unrelated AT-rich DNA probes (B). C. AT-rich DNA probe containing a single AT-run (AT-3) and associated control probe (Ctrl-3). D. DNA pulldown with AT-rich (AT-1, AT-2, AT-3) or control (Ctrl-1, Ctrl-2, Ctrl-3) probes followed by Western blot analysis for SALL4 using WT ESC protein extracts. E. Protein alignment and consensus sequence of C2H2 zinc-finger cluster 4 (ZFC4) in the mouse SALL protein family. ZFC4 is absent in SALL2. F. Western blot quantification of SALL4 expression levels in S4KO and ZFC4mut/ Δ ESCs, normalised to HDAC1 expression and relative to WT ESC levels. Data points indicate independent replicate experiments and error bars standard deviation. G. SALL4 co-immunoprecipitation with SALL1 and NuRD components in WT, S4KO (negative control) and ZFC4mut/ Δ ESCs.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Mass Spectrometry, Western Blot, Sequencing, Expressing, Standard Deviation, Immunoprecipitation, Negative Control

    A. Statistical analysis of AT-dependent gene expression changes (coefficient estimates with 99% confidence intervals) observed with ZFC4-regulated genes (see Figure 3A ). Significance is attributed by the F-test. Empty circles represent non-significant model fits ( > 0.01 FDR) and filled circles represent a significant fit to the model. B. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-independent genes (see Figure 3A ) divided into five equal categories according to AT-content. C. Statistical analysis of AT-dependent gene expression changes observed with ZFC4-independent genes, as described in panel A. D. RT-qPCR analysis following 48h doxycycline induction in the indicated ESC lines (see Figure 3E ), or in WT and S4KO control ESCs. Sall4 mRNA expression was normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation. E. SALL4 immunofluorescence following 48h doxycycline induction in the indicated ESC lines (see Figure 3E ), or in WT and S4KO control ESCs. DNA was stained with DAPI. Scale bars: 100μm. F. Scatter plot showing the relative expression of genes deregulated both in S4KO ESCs and following SALL4 re-expression. G. Profile plot showing the density of A/T nucleotides around the transcription unit of Sall4-responsive genes (see Figure 3F ) divided into five equal categories according to AT-content. H, I. Statistical analysis of AT-dependent gene expression changes observed with Sall4-responsive (H) and ZFC4-regulated (I) genes, as described in panel A. J. Profile plot showing the density of A/T nucleotides around the transcription unit of EGFP-responsive genes (see Figure 3F ) divided into five equal categories according to AT-content. K. Correlation between EGFP-induced gene expression changes and DNA base composition. EGFP-responsive genes were divided into five equal categories depending on their AT-content, and their relative expression levels were analysed in the indicated ESC lines. L. Statistical analysis of AT-dependent gene expression changes observed with EGFP-responsive genes, as described in panel A. M. Profile plot showing the density of A/T nucleotides around the transcription unit of SALL4-independent genes changing during early ESC differentiation (see Figure 3J ) divided into five equal categories according to AT-content. N. Statistical analysis of AT-dependent gene expression changes observed with SALL4-independent genes (light blue), SALL4-dependent genes controlled by ZFC4 (red) and SALL4-dependent genes not controlled by ZFC4 (grey) during early differentiation of WT cells (day 0 vs day 2), as described in panel A.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: A. Statistical analysis of AT-dependent gene expression changes (coefficient estimates with 99% confidence intervals) observed with ZFC4-regulated genes (see Figure 3A ). Significance is attributed by the F-test. Empty circles represent non-significant model fits ( > 0.01 FDR) and filled circles represent a significant fit to the model. B. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-independent genes (see Figure 3A ) divided into five equal categories according to AT-content. C. Statistical analysis of AT-dependent gene expression changes observed with ZFC4-independent genes, as described in panel A. D. RT-qPCR analysis following 48h doxycycline induction in the indicated ESC lines (see Figure 3E ), or in WT and S4KO control ESCs. Sall4 mRNA expression was normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation. E. SALL4 immunofluorescence following 48h doxycycline induction in the indicated ESC lines (see Figure 3E ), or in WT and S4KO control ESCs. DNA was stained with DAPI. Scale bars: 100μm. F. Scatter plot showing the relative expression of genes deregulated both in S4KO ESCs and following SALL4 re-expression. G. Profile plot showing the density of A/T nucleotides around the transcription unit of Sall4-responsive genes (see Figure 3F ) divided into five equal categories according to AT-content. H, I. Statistical analysis of AT-dependent gene expression changes observed with Sall4-responsive (H) and ZFC4-regulated (I) genes, as described in panel A. J. Profile plot showing the density of A/T nucleotides around the transcription unit of EGFP-responsive genes (see Figure 3F ) divided into five equal categories according to AT-content. K. Correlation between EGFP-induced gene expression changes and DNA base composition. EGFP-responsive genes were divided into five equal categories depending on their AT-content, and their relative expression levels were analysed in the indicated ESC lines. L. Statistical analysis of AT-dependent gene expression changes observed with EGFP-responsive genes, as described in panel A. M. Profile plot showing the density of A/T nucleotides around the transcription unit of SALL4-independent genes changing during early ESC differentiation (see Figure 3J ) divided into five equal categories according to AT-content. N. Statistical analysis of AT-dependent gene expression changes observed with SALL4-independent genes (light blue), SALL4-dependent genes controlled by ZFC4 (red) and SALL4-dependent genes not controlled by ZFC4 (grey) during early differentiation of WT cells (day 0 vs day 2), as described in panel A.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Immunofluorescence, Staining

    A. Western blot analysis of SALL4 in WT, ZFC4mut heterozygote (Het) and homozygote ( Hom ) embryos at E10.5. WT and S4KO ESC protein extracts were used as controls. B. Western blot quantification of SALL4 expression levels in ZFC4mut embryos (as presented in panel A), normalised to Histone H3 expression and relative to WT . Data points indicate independent embryos and error bars standard deviation.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: A. Western blot analysis of SALL4 in WT, ZFC4mut heterozygote (Het) and homozygote ( Hom ) embryos at E10.5. WT and S4KO ESC protein extracts were used as controls. B. Western blot quantification of SALL4 expression levels in ZFC4mut embryos (as presented in panel A), normalised to Histone H3 expression and relative to WT . Data points indicate independent embryos and error bars standard deviation.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Western Blot, Expressing, Standard Deviation

    A. DNA pulldown with AT-rich probes containing all possible combinations of AT 5 mers or control probes with disrupted AT-runs (Ctrl-) followed by Western blot analysis for SALL4. Amounts of DNA probes were assessed by agarose gel analysis and SALL4 enrichment was normalised to input. Data points indicate independent replicate experiments and error bars standard deviation. B. Detection of non-specific SALL4 ChIP-seq peaks in Sall4 knockout ESCs (negative control) using either a monoclonal or a polyclonal anti-SALL4 antibody. C. Profile plot and heatmap showing SALL4 ChIP-seq signal in Sall4 knockout ESCs at non-specific sites (see panel B) using either a monoclonal or a polyclonal anti-SALL4 antibody. D. Venn diagrams showing the overlap of SALL4 ChIP-seq peaks between independent replicate experiments using an anti-SALL4 monoclonal antibody in WT (blue) and ZFC4mut (red) ESC lines. E. Profile plots and heatmaps showing SALL4, H3K4me1 and H3K27ac ChIP-seq signal at SALL4 WT ChIP-seq peaks in WT ESCs.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: A. DNA pulldown with AT-rich probes containing all possible combinations of AT 5 mers or control probes with disrupted AT-runs (Ctrl-) followed by Western blot analysis for SALL4. Amounts of DNA probes were assessed by agarose gel analysis and SALL4 enrichment was normalised to input. Data points indicate independent replicate experiments and error bars standard deviation. B. Detection of non-specific SALL4 ChIP-seq peaks in Sall4 knockout ESCs (negative control) using either a monoclonal or a polyclonal anti-SALL4 antibody. C. Profile plot and heatmap showing SALL4 ChIP-seq signal in Sall4 knockout ESCs at non-specific sites (see panel B) using either a monoclonal or a polyclonal anti-SALL4 antibody. D. Venn diagrams showing the overlap of SALL4 ChIP-seq peaks between independent replicate experiments using an anti-SALL4 monoclonal antibody in WT (blue) and ZFC4mut (red) ESC lines. E. Profile plots and heatmaps showing SALL4, H3K4me1 and H3K27ac ChIP-seq signal at SALL4 WT ChIP-seq peaks in WT ESCs.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Western Blot, Agarose Gel Electrophoresis, Standard Deviation, Chromatin Immunoprecipitation, Knock-Out, Negative Control

    Phenotypic characterisation of SALL4 ZFC4 mutation during neuronal differentiation A. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm. B. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: Phenotypic characterisation of SALL4 ZFC4 mutation during neuronal differentiation A. TUJ1 immunofluorescence in the indicated ESC lines cultured in serum/LIF medium, and following differentiation for 5 days in N2B27 medium. DNA was stained with DAPI. Scale bars: 100μm. B. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Mutagenesis, Immunofluorescence, Cell Culture, Staining, Quantitative RT-PCR, Standard Deviation

    A. SALL4 co-immunoprecipitation with SALL1 and NuRD components in WT, S4KO (negative control) and ZFC1-2 Δ ESCs. B. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between independent replicate experiments in ZFC1-2 Δ ESCs. C. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between WT, ZFC1-2 Δ and ZFC4mut ESCs. D, E. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-regulated (D) and ZFC1/2-regulated (E) genes (see Figure 6E ) divided into five equal categories according to AT-content. F. Statistical analysis of AT-dependent gene expression changes (coefficient estimates with 99% confidence intervals) observed with ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes (see Figure 6E ). Significance is attributed by F-test. Empty circles represent non-significant model fits ( > 0.01 FDR) and filled circles represent significant model fit. G. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: A. SALL4 co-immunoprecipitation with SALL1 and NuRD components in WT, S4KO (negative control) and ZFC1-2 Δ ESCs. B. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between independent replicate experiments in ZFC1-2 Δ ESCs. C. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between WT, ZFC1-2 Δ and ZFC4mut ESCs. D, E. Profile plot showing the density of A/T nucleotides around the transcription unit of ZFC4-regulated (D) and ZFC1/2-regulated (E) genes (see Figure 6E ) divided into five equal categories according to AT-content. F. Statistical analysis of AT-dependent gene expression changes (coefficient estimates with 99% confidence intervals) observed with ZFC4-regulated (red) and ZFC1/2-regulated (purple) genes (see Figure 6E ). Significance is attributed by F-test. Empty circles represent non-significant model fits ( > 0.01 FDR) and filled circles represent significant model fit. G. RT-qPCR analysis of the neuronal markers Tuj1, Ascl1 and Nestin in the indicated cell lines following differentiation for 5 days in N2B27 medium. Transcripts levels were normalised to TBP and expressed relative to WT . Data points indicate independent replicate experiments and error bars standard deviation.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Immunoprecipitation, Negative Control, Chromatin Immunoprecipitation, Expressing, Quantitative RT-PCR, Standard Deviation

    Phenotypic characterisation of SALL4 ZFC4 mutation during embryonic development A. Table showing the number of live pups and embryos at different stages of development, and their associated genotype. Animals were crossed to obtain ZFC4mut heterozygous ( Het ), homozygous ( Hom ), or WT progeny. B. Diagram showing the results from crossing ZFC4mut heterozygote mice. ZFC4mut homozygous animals die during embryonic development. C. Representative images of WT, ZFC4mut heterozygous (Het) and homozygous ( Hom ) embryos at E10.5, taken at the same magnification.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: Phenotypic characterisation of SALL4 ZFC4 mutation during embryonic development A. Table showing the number of live pups and embryos at different stages of development, and their associated genotype. Animals were crossed to obtain ZFC4mut heterozygous ( Het ), homozygous ( Hom ), or WT progeny. B. Diagram showing the results from crossing ZFC4mut heterozygote mice. ZFC4mut homozygous animals die during embryonic development. C. Representative images of WT, ZFC4mut heterozygous (Het) and homozygous ( Hom ) embryos at E10.5, taken at the same magnification.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Mutagenesis, Mouse Assay

    Characterisation of SALL4 C2H2 zinc-finger cluster 4 (ZFC4) DNA binding in vitro and in vivo A. SALL4 ZFC4 protein fragment used for in vitro HT-SELEX experiments. B. Preferred 5-mer motif identified after 6 cycles of HT-SELEX with SALL4 ZFC4. C. Relative enrichment of 5-mer motifs categorised by AT-content at cycle 0, 3 and 6 of HT-SELEX with SALL4 ZFC4. Coloured circles are the most enriched motifs at cycle 6 of HT-SELEX. D. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between WT and ZFC4mut ESCs. E. Profile plot and heatmap showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. F. Venn diagram showing the overlap of SALL4 ChIP-seq peaks detected in WT ESCs with ATAC-seq peaks (accessible chromatin) and CpG islands. G. Results from de novo motif analysis at SALL4 WT ChIP-seq peaks (summit +/- 250bp) showing the relative frequency of each DNA motif and its associated E-value. ATAC-seq peaks were used as a control for regions of accessible chromatin. H. Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit +/- 250bp) in WT (blue) and ZFC4mut (red) ESCs. CpG islands and ATAC-seq peaks coincide with regions of accessible chromatin and are shown for comparison.

    Journal: bioRxiv

    Article Title: SALL4 controls cell fate in response to DNA base composition

    doi: 10.1101/2020.06.30.179481

    Figure Lengend Snippet: Characterisation of SALL4 C2H2 zinc-finger cluster 4 (ZFC4) DNA binding in vitro and in vivo A. SALL4 ZFC4 protein fragment used for in vitro HT-SELEX experiments. B. Preferred 5-mer motif identified after 6 cycles of HT-SELEX with SALL4 ZFC4. C. Relative enrichment of 5-mer motifs categorised by AT-content at cycle 0, 3 and 6 of HT-SELEX with SALL4 ZFC4. Coloured circles are the most enriched motifs at cycle 6 of HT-SELEX. D. Venn diagram showing the overlap of SALL4 ChIP-seq peaks between WT and ZFC4mut ESCs. E. Profile plot and heatmap showing SALL4 ChIP-seq signal at SALL4 WT ChIP-seq peaks in the indicated cell lines. F. Venn diagram showing the overlap of SALL4 ChIP-seq peaks detected in WT ESCs with ATAC-seq peaks (accessible chromatin) and CpG islands. G. Results from de novo motif analysis at SALL4 WT ChIP-seq peaks (summit +/- 250bp) showing the relative frequency of each DNA motif and its associated E-value. ATAC-seq peaks were used as a control for regions of accessible chromatin. H. Analysis of the DNA base composition surrounding SALL4 ChIP-seq peaks (summit +/- 250bp) in WT (blue) and ZFC4mut (red) ESCs. CpG islands and ATAC-seq peaks coincide with regions of accessible chromatin and are shown for comparison.

    Article Snippet: For each immunoprecipitation, 700μg of chromatin was mixed with 5μg of anti-SALL4 antibody (Santa Cruz ref. sc-101147, RRID:AB_1129262 or Abcam ref. ab29112, RRID:AB_777810) in a total volume of 1ml of sonication buffer supplemented with 1x protease inhibitor cocktail.

    Techniques: Binding Assay, In Vitro, In Vivo, Chromatin Immunoprecipitation