Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Phos-tag analyses reveal that DNA Top2 Ser 1363 and Ser 1364 in the C-terminal charged region are phosphorylated. A , schematic of the fission yeast Top2 polypeptide. Top2 consists of a catalytic core and acidic/basic clusters at both ends of the protein. The catalytic core contains two evolutionarily conserved domains, an ATP-binding hydrolytic domain ( green ), and a DNA-binding cleavage domain ( red ). B , Phos-tag analysis of FLAG-tagged Top2 protein. Extracts of a cs nda3-KM311 β-tubulin mutant strain expressing Top2–3FLAG were prepared from asynchronously cultured ( AS ) and mitotically arrested cells ( M ) and run on SDS-PAGE gels in the presence or absence of 25 μ m Phos-tag. The untagged strain was used as a negative control. Each sample was preincubated with phage λ PPase (+) or buffer (−). Ponceau staining served as a loading control. Anti-FLAG antibodies detected phosphorylated Top2 proteins ( Top2-P ) in asynchronous and mitotically arrested cells. The position of the marker band is not indicated in the phos-tag blot because the marker proteins were also highly phosphorylated, and precise positions were unclear. C , Phos-tag analysis of truncated Top2 proteins. Top panel , N- or C-terminally truncated Top2-FLAG proteins were mildly overproduced under the inducible nmt promoter (plasmid Rep81) in the absence of thiamine in WT S. pombe cells. A strain containing only the vector was used as a control. Positions of unphosphorylated bands are indicated by red arrowheads. Bottom panel , truncated fragments and their phosphorylation ( Phospho ) status are indicated (+, phosphorylated; −, unphosphorylated). A red dashed box shows the predicted region of phosphorylation. aa , amino acids. D , C-terminally truncated Top2 proteins were expressed under the native promoter with the chromosomally integrated FLAG-tagged gene. Top2 C terminus phosphorylation is predicted between residues 1352 and 1391 ( double-headed arrow in the bottom panel ). E , the amino acid sequence around Ser 1363 and Ser 1364 of S. pombe Top2, with seven-amino-acid sequences. The consensus target sequence for CKII is shown. Xl , Xenopus laevis ; Hs , Homo sapiens ; Dm , Drosophila melanogaster ; Ce , Caenorhabditis elegans ; At , Arabidopsis thaliana ; Sp , Schizosaccharomyces pombe ; Sc , Saccharomyces cerevisiae. F , the Phos-tag–mediated mobility shift of the Top2 protein disappeared as a result of alanine substitutions for Ser 1363 and Ser 1364 . The intensity of the smeary phosphorylated Top2 bands was quantified and is indicated relative to the background.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Binding Assay, Mutagenesis, Expressing, Cell Culture, SDS Page, Negative Control, Staining, Marker, Plasmid Preparation, Sequencing, Mobility Shift

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Top2 Ser 1363 and Ser 1364 are phosphorylated by CKII throughout the cell cycle. A and B , the specificity of polyclonal antibodies against phospho-Ser 1363 –containing ( A ) and phospho-Ser 1364 –containing ( B ) phosphopeptides was examined. Asynchronous extracts of the strain expressing FLAG-tagged WT Top2 protein or nonphosphorylatable alanine mutants of Top2 (S1363A or S1364A) proteins were prepared and run on SDS-PAGE. Ponceau staining was used for the loading control of extracts. C , Ser 1363 and Ser 1364 were phosphorylated by CKII in vitro . Immunoprecipitated Top2-FLAG proteins were dephosphorylated by λ protein phosphatase and then incubated with CKII. Anti-Top2 phospho-Ser 1363 and -phospho-Ser 1364 antibodies were used to detect rephosphorylation by CKII. D , phosphorylation of Ser 1363 and Ser 1364 was diminished in two distinct alleles of CKII ts mutants, cka1-372 and orb5/cka1-19 , at the restrictive temperature (36 °C for 6 h). Cell extracts were prepared in WT, cka1-372 , orb5/cka1-19 and tor2-S mutants expressing Top2-FLAG protein at 26 °C and 36 °C (6 h) along with the untagged strain. The tor2-S mutant was used as a control strain , which shows small cells as observed in cka1-372 and cka1/orb5-19 mutant cells at the restrictive temperature ( Fig. S3 A ). Top2 proteins were detected with anti-FLAG antibodies in the presence ( Phos-tag ) or absence ( normal ) of 25 μ m Phos-tag. Top2 Ser 1363 and Ser 1364 phosphorylation was detected using anti-phospho-Ser 1363 and anti-phospho-Ser 1364 antibodies, respectively. Anti-PSTAIR (Cdc2) antibody was used for a loading control of extracts. The asterisk indicates nonspecific bands that probably appear under delay or arrest of cell-cycle progression, such as under nitrogen starvation ( F ), UV irradiation ( Fig. S5 ), and low-glucose conditions ( Figs. S4 A and S5 ). Because FLAG tagging partly reduces the Top2 protein level ( Fig. S4 B ), both phospho-specific antibodies give a weaker signal in FLAG-tagged strains relative to untagged strains. E , Ser 1363 and Ser 1364 were phosphorylated throughout the cell cycle. Block and release of cdc25-22 mutant cells expressing Top2–3FLAG was done for synchronous culture commencing from late G 2 phase to mitosis. Immunoblotting was performed with antibodies against FLAG, Top2 phospho-Ser 1363 and phospho-Ser 1364 . Cut2 (securin) and Cdc13 (mitotic cyclin) are shown as mitotic progression markers. Cell cycle progression was monitored by counting the number of binucleate cells lacking ( blue , anaphase–telophase) and possessing septa ( red , G 1 /S phase). F , top panel , Top2 phosphorylation was examined in nitrogen-starved, WT, G 0 -arrested cells, which were then permitted to proliferate by addition of a nitrogen source . Immunoblotting was performed with antibodies against FLAG, Top2 phospho-Ser 1363 , and tubulin (a loading control) as shown in D. Bottom panel , FACScan analysis indicating the timing of S phase (5–6 h). Top2 phosphorylation did not change during nitrogen starvation or after release.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Expressing, SDS Page, Staining, In Vitro, Immunoprecipitation, Incubation, Mutagenesis, Irradiation, Blocking Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Top2–2A (S1363A,S1364A) mutant protein maintains decatenation activity but has reduced ATPase activity. A , SDS-PAGE patterns of WT Top2 and alanine substitution mutant Top2-2A (S1363A and S1364A). FLAG-tagged Top2-WT and -2A proteins were overproduced under the inducible nmt promoter (plasmid Rep41) in WT S. pombe cells and immunoprecipitated using anti-FLAG antibody. A strain containing only the vector was used as a control. Immunoprecipitated FLAG-tagged Top2 proteins are indicated by arrows . The position of the protein marker bands ( M ) is indicated. B , Top2 decatenation assay. kDNA (153 ng) was incubated with immunoprecipitated Top2 fractions in ATP-containing reaction buffer (see “Experimental procedures”) for 1–30 min at 37 °C. An immunoprecipitated fraction from cell extracts containing only the empty vector was used for a mock reaction ( vector IP ). The reaction was terminated using stop buffer and loaded onto a 1% agarose gel followed by ethidium bromide staining. Only the catenated kDNA ( kDNA ) and decatenated kDNA ( decat. kDNA ) were loaded as controls, along with λDNA digested by EcoT14I (λ -EcoT14I ). Positions of catenated and decatenated kDNA are indicated by an arrow and a vertical line , respectively. The ratio of decatenated DNA to total DNA (catenated + decatenated) was quantified. Phosphorylation of Ser 1363 and Ser 1364 does not affect Top2 decatenation activity. C , Top2 ATPase assay. Immunoprecipitated Top2-WT or -2A mutant proteins were incubated with ATP and kDNA in the presence or absence of the 5 μ m anti-cancer topo II inhibitor ICRF-193 for 30 min at 30 °C. Free phosphate produced by ATP hydrolysis was measured by malachite green colorimetric reagent (see “Experimental procedures”). Error bars represent the standard deviation for each experiment performed in triplicate. p values for comparison among four conditions were calculated using one-way analysis of variance with Holm multi-comparison correction. *, p < 0.05.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Mutagenesis, Activity Assay, SDS Page, Plasmid Preparation, Immunoprecipitation, Marker, Incubation, Agarose Gel Electrophoresis, Staining, ATPase Assay, Produced, Standard Deviation

Journal: The Journal of Biological Chemistry

Article Title: Casein kinase II–dependent phosphorylation of DNA topoisomerase II suppresses the effect of a catalytic topo II inhibitor, ICRF-193, in fission yeast

doi: 10.1074/jbc.RA118.004955

Figure Lengend Snippet: Defective chromosome segregation induced by an anti-cancer catalytic topo II inhibitor, ICRF-193, is exacerbated in cka1-372 and top2-2A (S1363A,S1364A) mutant cells. A , CKII ts mutant cka1-372 cells showed more severe defects in mitotic chromosome segregation than WT cells in the presence of ICRF-193. Left panel , DAPI-stained micrographs of WT and cka1-372 mutant cells were obtained at the restrictive temperature (36 °C) for 3 h in the presence of DMSO or ICRF-193 (5 and 10 μ m ). A displaced nuclear phenotype was frequently observed in ICRF-193–treated cka1 mutant cells ( arrows ). Right panel , frequencies of defective phenotypes categorized as lagging-like ( blue ), streaked chromosomes ( red ), and displaced nucleus ( green ). More than 200 anaphase cells were counted for each sample. Error bars represent the standard deviation for each defective phenotypes. p values for comparison between the drug-treated WT and cka1 mutant were calculated using a Student's t test. *, p < 0.05; **, p < 0.01 ( black , total frequency of abnormal phenotypes; color , each phenotype). B , defects in chromosome segregation increased significantly in unphosphorylatable top2-2A mutants compared with WT and phosphomimetic top2–2E mutant cells in the presence of ICRF-193. Cells were asynchronously cultured at 26 °C for 2 h in the presence of DMSO or 5 μ m ICRF-193. Left panel , representative micrographs of DMSO- and 5 μ m ICRF-193–treated top2-2A cells. Chromatin DNA was stained with DAPI. Abnormally streaked chromosomes in anaphase are indicated ( arrows ). Scale bar = 10 μm. Right panel , frequencies of anaphase cells with abnormally streaked chromosomes. More than 200 anaphase cells were counted for each sample. Error bars represent the standard deviation for each experiment performed in biological triplicates. Significant differences among the three strains were examined using one-way analysis of variance with Bonferroni multi-comparison correction. *, p < 0.05; **, p < 0.01; n.s. , not significant.

Article Snippet: Dephosphorylated Top2 proteins were incubated with 500 units of casein kinase II protein complex (α and β subunits) derived from human cells (New England Biolabs, P6010) in the presence of 200 μ m ATP for 30 min at 30 °C.

Techniques: Mutagenesis, Staining, Standard Deviation, Cell Culture

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Hela, HepG2, and Huh7 cells were seeded in a 12-well plate for overnight, then left untreated or treated with human IFN-α (1,000 IU/ml) for 18 or 36 h. The untreated cells (control) were harvested together with the cells treated by IFN-α for 36 h. ISG20 expression was detected by Western blot. Hela cells transfected with plasmid F-ISG20 expressing the FLAG-tagged ISG20 were used as positive control for ISG20 Western blot (lane 2). β-actin expression was presented as loading control. (B) PHHs and HepG2 cells were cultured in 12-well-plate and treated with type I IFN-α (1,000 IU/ml), type II IFN-γ (100 ng/ml) or type III IFN-λ (100 ng/ml), or left untreated (control) for 2 days. The levels of ISG20 expression were determined by Western blot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Control, Expressing, Western Blot, Transfection, Plasmid Preparation, Positive Control, Cell Culture

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with either pHBV1.3 and F-ISG20 or empty vector, or pCMVHBV and F-ISG20 or empty vector, as indicated. Cells were harvested at day 5 post-transfection, and the levels of viral RNA and DNA were determined by Northern (top) and Southern (middle) blot hybridization, respectively. For RNA analysis, each lane was loaded with 10 μg of total RNA and probed with a genome-length, plus-strand-specific HBV riboprobe. Ribosomal RNAs (28S and 18S) are presented as loading controls. The positions of HBV pgRNA (3.5kb) and subgenomic surface RNAs (2.4kb and 2.1kb) are indicated. For DNA analysis, HBV core DNA was probed with genome-length, minus-strand-specific HBV riboprobe. The positions of relaxed circular (RC) and single-stranded (SS) DNAs are indicated. The relative pgRNA, sRNA or total DNA replicative intermediate level in each sample is expressed as the percentage of RNA or DNA of the cells transfected with empty vector. ISG20 overexpression was confirmed by Western blot using monoclonal antibodies against FLAG-tag. β-actin expression was presented as protein loading control (bottom panels). (B) The same experiment was done in Huh7 cells with pHBV1.3 as HBV expression vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Northern Blot, Hybridization, Over Expression, Western Blot, Bioprocessing, FLAG-tag, Expressing, Control

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) HepDES19 cells were seeded in 35 mm-dish and cultured with tet-free medium to induce HBV pgRNA transcription. 24 h later, cells were transfected with 4 μg of control vector or plasmid F-ISG20 for 36 h, then tet was added back to the culture medium to shut down pgRNA transcription. Cells were harvested at indicated time points. HBV RNA was extracted from harvested samples and analyzed by Northern blot. Expression of FLAG-tagged ISG20 was detected by Western blot. The results are representative of three separate trials. (B) HepG2 cells in 12-well-plate were co-transfected with 0.7 μg of pTREHBVDES and 0.1 μg of pTet-off, plus 0.7 μg of control vector or plasmid F-ISG20. Four days post transfection, tet was added back and cells were harvested at indicated time points and subjected to HBV RNA qPCR analysis. The relative levels of HBV total RNA normalized to β-actin mRNA levels in each samples were expressed as the percentage of the RNA levels from the corresponding sample at 0 h time point (Mean ± SD, n = 4). The half-life of HBV RNA was marked on the plot.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Cell Culture, Transfection, Control, Plasmid Preparation, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepDES19 cells were transfected with 100 nM of control siRNA (Lane 1–3) or ISG20 siRNA (siISG20) (lane 4–6) twice with a 24 h interval after tet being withdrawn. Culture medium was replaced 12 h after the 2nd siRNA transfection, and cells were either left untreated as controls (lane 1 &4) or treated with 100 IU/ml (lanes 2 & 5) or 1,000 IU/ml (lanes 3 & 6) of IFN-α. Cells were harvested 5 days after 2nd transfection. Viral total RNA (top panel), encapsidated pgRNA (upper middle panel), and core DNA (lower middle panel) were subjected to Northern and Southern analyses, respectively. ISG20 protein expression was revealed by Western blot, and β-actin served as loading control (bottom panels). The relative levels of viral nucleic acids and ISG20 expression in the siISG20 transfected or IFN-α treated samples (lanes 2–6) are expressed as the percentage of the control sample (lane 1). The data presented here are representative of two independent experiments.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Northern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2-NTCP12 cells stably transduced by control lentiviral shRNA (shcontrol) or ISG20 lentiviral shRNA (shISG20) were spinoculated with HBV at 100 vge/cell. 16 h later, the infected cells were mock treated or treated with 1,000 IU/ml of IFN-α for 6 days, and the cells were subjected to the following analyses: (A) The expression of ISG20 was analyzed by Western blot. (B) HBV infectivity was assessed by HBcAg immunofluorescence, and the percentage of HBcAg-positive cells were calculated from multiple microscopic field of view (mean±SD, n = 5). Nuclei were stained with DAPI. (C) HBV total RNA were quantified by qPCR and the relative expression levels to β-actin mRNA were plotted as fold change to control samples (HBV infected shcontrol cells without IFN-α treatment) (mean±SD, n = 3).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Stable Transfection, Control, shRNA, Infection, Expressing, Western Blot, Immunofluorescence, Staining

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were transfected with pHBV1.3 and equal amount of control vector (lanes 1 & 2) or F-ISG20 (lanes 3 & 4) or F-ISG20 D94G (lanes 5 & 6). Cells were harvested 5 days post-transfection and levels of HBV RNA (1st panel from the top) and encapsidated pgRNA (4th panel from the top) were determined by Northern blot hybridization. The assembled HBV capsid was revealed by native capsid gel EIA assay (3rd panel from the top) and the viral DNA in capsid was detected in situ by hybridization (5th panel from the top). HBV core DNA replicative intermediates were extracted and analyzed by Southern blot (6th panel from the top). Expression of FLAG-tagged ISG20 proteins was revealed by Western blot and β-actin served as loading control (bottom two panels). Results from duplicate experiments are presented.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Hybridization, Enzyme Immunoassay, In Situ, Southern Blot, Expressing, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells were co-transfected with plasmid pCMVHBVΔCΔP and either control vector (lane 1) or FLAG-Pol (lanes 2&3), or pCMVHBV with either control vector (lane 4) or F-ISG20 D94G (lanes 5&6). Cells were harvested 4 days post-transfection. Input HBV RNA was determined by Northern blot (top panels). Input FLAG-Pol and F-ISG20 D94G proteins were determined by Western blot using FLAG Ab (top panels). Cell lysates were immunoprecipitated with beads coated with FLAG Ab, the immunoprecipitated Pol and ISG20 D94G were revealed by Western blot using FLAG Ab (lanes 3&6, bottom panel), and the bound RNA was extracted by Trizol and analyzed by Northern blot (lanes 3&6, bottom panel). HA Ab pull-down served as negative controls (lanes 2 & 5, bottom panel). See for more experimental details.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Plasmid Preparation, Control, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: HepG2 cells in 6-well-plate were co-transfected with 1 μg of pCMVHBVΔCΔP and 5 μg control vector (lane 1) or 1 μg of FLAG-Pol in the absence of HA-ISG20 D94G (lane 2) or increased amount of HA-ISG20 D94G (1 μg, 2 μg, 4 μg; lanes 3–5). The total amount of transfected DNA was kept constant (6 μg/well) by adding control vector plasmid (lanes 2–4). 5 days later, total cellular HBV RNA and protein (FLAG-Pol and HA-ISG20 D94G ) were determined by Northern and Western blot, respectively, as input controls (top panels). Immunoprecipitation was performed by using antibodies against HA or FLAG epitopes, followed by Northern blot analysis of HBV RNA (bottom panels).

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Transfection, Control, Plasmid Preparation, Northern Blot, Western Blot, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of HBV pgRNA deletion clones. Plasmid pHBV1.3 contains a 1.3 overlength HBV genome (Genbank Accession Number U95551), starting at nt 1000. The HBV nucleotide positions are according to Galibert et al . Cp represents the HBV core promoter. pA is the polyadenylation site. The arrow indicates the pgRNA transcription initiation site (nt 1820). Three major HBV mRNA (3.5 kb, 2.4 kb, and 2.1 kb) are depicted underneath the 1.3 mer HBV DNA template. The solid dot indicates 5’ cap of mRNA; and the sawtooth line represents the polyA tail at the 3’ terminus of mRNA. The internal deletion clones (pg-IDs) are described in details in . The terminal redundancy (TR) deletion clones contain truncations of HBV sequences (nt 1820–1918) at either 5’ or 3’ terminus of pgRNA coding sequences (pg-Δ5TR and pg-Δ3TR, respectively.), or both (pg-Δ5/3TR). The transcription of terminal truncated pgRNA is governed by CMV-IE promoter in the pCDNA3.1/V5-His-TOPO vector. (B) Sensitivity of HBV RNA with TR deletion to ISG20-mediated RNA reduction. HepG2 cells were transfected with HBV TR deletion clone and control plasmid or F-ISG20 plasmid. Cells were harvested at day 4 post transfection and subjected to viral RNA analysis by Northern hybridization. ( C) HBV TR insertion renders Luc gene to be sensitive to ISG20. The schematic illustration indicates the reporter construct EnII/Cp-Luc with HBV TR insertion at the flanking non-translational region of luciferase ORF. HepG2 cells were transfected with each indicated reporter plasmid and control vector or plasmid expressing ISG20. Cells were lysed at day 3 post transfection and luciferase activity was measured. The plotted relative luciferase activity (RLA) represents the mean ± SD (n = 3) of the percentage of absorbance obtained from wells transfected with ISG20 over control vector.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Clone Assay, Plasmid Preparation, Transfection, Control, Northern Blot, Hybridization, Construct, Luciferase, Expressing, Activity Assay

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic stem-loop structure of HBV ε RNA. Ribonucleotide sequences (nt 1847–1991, genotype D, subtype ayw) are presented with base paring indicated by dotted line. (B) Verification of the purified recombinant 6×His-tagged ISG20 by SDS-PAGE Coomassie staining. (C) EMSA assay of ISG20-ε binding. The indicated amount of ISG20 proteins were incubated with 100 ng 32 P-end-labeled ε RNA in binding buffer to form nucleoprotein complexes. Monoclonal anti-His antibody was used for supershifting of the His-ISG20/ HBV ε complex. Excessive amount of cold unlabeled HBV ε RNA (10×, 20×, 40×) were used to compete with the binding of ISG20 to 100 ng radiolabeled HBV ε. The nucleoprotein complexes were separated by native PAGE and the shifted bands were detected by autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Purification, Recombinant, SDS Page, Staining, Binding Assay, Incubation, Labeling, Clear Native PAGE, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustrations of the wildtype HBV ε RNA and mutants. The substructural domains of ε, including the lower stem, bulge, upper stem, and apical loop, are marked on the full-length form. Shorter versions of ε include the upper stem loop (US+L), lower stem with wildtype or mutant bulge sequence as loop (LS+B, LS+Bm), and LS+B with bottom 4 base-pairs removed from the lower stem (LSΔ4+B). These RNA fragments were chemically synthesized and 5’ end radiolabeled for ISG20 EMSA. (B) EMSA of ISG20 binding with full-length (FL) ε, US+L, and LS+B. (C) EMSA of ISG20 binding with LS+B, LS+Bm, and LS+B. (D) HepG2 cells were transfected with plasmid pMS transcribing the 2.1kb HBV RNA, or pMSΔ4bp transcribing the 2.1kb HBV with 4 nucleotides removed from the bottom right arm of the lower stem of ε, in the absence or presence of F-ISG20. HBV RNA and FLAG-tagged ISG20 were analyzed by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Mutagenesis, Sequencing, Synthesized, Binding Assay, Transfection, Plasmid Preparation, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: (A) Schematic illustration of ISG20. The amino acid (a.a) positions are labeled with numbers. The gray boxes indicate the predicted Exo motifs. The enzymatic mutant site (D94G) is marked with an asterisk. (B) Bacterially expressed His-tagged ISG20 and mutants were purified and examined by SDS-PAGE Coomassie staining. The asterisk indicates a nonspecific protein band co-purified with the recombinant ΔExoII mutant. (C) EMSA of ε binding by wildtype ISG20 and the indicated mutants. (D) HepG2 cells were co-transfected with pHBV1.3 and control vector or indicated FLAG-ISG20 constructs. HBV RNA and ISG20 proteins were detected by Northern and Western blot, respectively.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Labeling, Mutagenesis, Purification, SDS Page, Staining, Recombinant, Binding Assay, Transfection, Control, Plasmid Preparation, Construct, Northern Blot, Western Blot

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: 0.1 µg of 5’-radiolabeled synthetic RNA substrates, specifically (A) the intact ε, upper stem-loop region (US+L), and lower stem with bulge serving as loop (LS+B); and (B) the intact ε, single-stranded left arm portion of ε, and 30-mer poly(rA), were incubated with the indicated amount of RNase A or purified His-ISG20 in nuclease reaction buffer for 15 min, then the reactions were terminated and the mixtures were fractionated through 10% TBE-Urea denaturing polyacrylamide gel, and the dried gel was subjected to autoradiography.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Incubation, Purification, Autoradiography

Journal: PLoS Pathogens

Article Title: Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA

doi: 10.1371/journal.ppat.1006296

Figure Lengend Snippet: The major viral intermediates and products at each major HBV replication steps are illustrated. cccDNA (or other HBV transcription template)-derived 3.5kb RNA (including precore mRNA and pgRNA) and other shorter subgenomic RNA species (2.4/2.1kb surface mRNA and 0.7kb X mRNA) are aligned to show the location of ε on different RNA species. The black circle dots indicate the 5’ cap of mRNA, the zigzag lines represent the polyA tails. ISG20 is shown as a rectangle box and its targeting sites on HBV RNA are indicated by arrowheads. As a consequence of ISG20-mediated HBV RNA degradation, the illustrations and labels of viral proteins/antigens, pgRNA encapsidation and DNA replication are shown in gray color schemes. The solid gray triangles indicate capsid proteins, viral polymerase is shown in an oval shape before ε binding and then a gray circle dot in the nucleocapsids after pgRNA encapsidation.

Article Snippet: Control siRNA and ISG20 siRNA were purchased from Santa Cruz Biotechnology for transient knock down experiments.

Techniques: Derivative Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Location of missense and nonsense mutations in SLC9A6/NHE6 variants of patients with Christianson syndrome. A, schematic planar drawing of the predicted membrane topology of the longest splice-variant of mammalian NHE6 and location of the mutations (yellow circles). Two consensus N-linked glycosylation sites (128NVT and 145NVS) within extracellular loop 2 have been verified experimentally (data not shown) and are illustrated in the drawing. B, phylogenetic comparison of the primary sequence of segments containing the various mutations in NHE6. The affected residues are shaded in black.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Variant Assay, Glycoproteomics, Comparison, Sequencing

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Structure homology modeling of transmembrane NHE6 variants associated with Christianson syndrome. A, front (membrane aspect, left panel) and top (extracellular aspect, right panel) views of a 3D structure homology model of dimeric human NHE6 based on the crystal structure of the bacterial T. thermophilus Na+/H+ antiporter NapA (TtNapA) (Protein Data Bank accession code 5bz3; 2.30 Å, 15% identity, 27% similarity), which provided the broadest coverage, highest resolution, and best spatial fit compared with other crystallized bacterial Na+/H+ antiporters. The proposed structure includes only the membrane-spanning helices (M2–M12; amino acids 74–540) that aligned with homologous segments of TtNapA. The top view includes the locations of the transmembrane-localized residues Leu-188 and Gly-383 mutated in CS. B, molecular dynamics simulation of structural changes predicted to occur in TM4 (highlighted in cyan) upon substitution of Leu-188 with Pro (L188P). The monomeric forms of NHE6 WT and L188P are illustrated. C and D, structural perturbations predicted to occur in the intramembranous re-entrant (R) loop between helices M8 and M9 upon substitution of Gly-383 with Asp (G383D). C, upper and lower panels show front and top views, respectively, of Gly-383, which is packed tightly against amino acids Phe-373, Ala-376, and Glu-377 (top left). Mutation of Gly-383 to Asp would result in steric clashes between these residues and interfere with the packing between these segments of the R-loop. D, G383D substitution would also disrupt interactions of R-loop residues Trp-379, Phe-381, and especially Thr-382 with residues Asp-92 and Ile-296 in helix M7 and residues Ile-330, Phe-331, and Ser-334 in helix M8.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Membrane, Mutagenesis

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the biosynthetic maturation of NHE6 variants. A, AP-1 cells transiently expressing NHE6GFP WT or CS-linked variants were lysed at the indicated time points (6–48 h) post-transfection. Equal amounts of proteins (20 μg) were subjected to SDS-PAGE and immunoblotting with a polyclonal anti-GFP antibody. NHE6 migrates as multiple bands: higher molecular weight bands represent the fully-glycosylated (fg) and core-glycosylated (cg) dimeric (d) forms of the exchanger that do not fully dissociate under SDS-PAGE conditions, whereas lower molecular weight bands represent fully-glycosylated and core-glycosylated forms of the dissociated monomeric (m) protein. The blots were stripped and reprobed with a mouse monoclonal anti-GAPDH antibody to control for protein loading. B, ratios of fully-glycosylated protein (monomer and dimer)/total NHE6 protein (fg/total) were quantified by densitometry of X-ray films exposed in the linear range and analyzed using ImageJ software. Values represent the mean ± S.D. of three different experiments. Significance was determined by two-way ANOVA with a post hoc Tukey test. The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Population means of the NHE6 variants are significantly different (F value = 26.4, p value = 1.5 × 10−16). Population means as a function of time are significantly different (F value = 48.6, p value = 1.9 × 10−19). § indicates significance (p < 0.01) of the means of NHE6 variants within a cluster relative to the 12-h time point. Asterisks indicate significance (★, p < 0.05, and ★★, p < 0.01) of the means between clusters of NHE6 variants at the indicated time points.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Expressing, Transfection, SDS Page, Western Blot, Molecular Weight, Control, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the protein stability of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT of CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points and lysed, and equal amounts of protein (20 μg) were analyzed by Western blotting using a mouse monoclonal anti-HA antibody. Blots were reprobed with a mouse monoclonal anti-GAPDH antibody to control for loading. Blots are representative images from four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of proteasomal and lysosomal inhibitors on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide (CHX) for the indicated time points in the presence of diluent (DMSO) and the proteasomal inhibitor MG-132 (40 μm) or the lysosomal inhibitor leupeptin/pepstatin (LeuP, 100 μg/ml). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of three separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Effect of the lysosomotropic agent chloroquine on cellular clearance of NHE6 variants. AP-1 cells were transiently transfected with NHE6HA WT or CS-linked variants for 24 h and then treated with 150 μg/ml cycloheximide for the indicated time points in the presence of diluent (H2O) or chloroquine (CQ, 500 μm). Total-cell lysates were analyzed by Western blotting with a mouse monoclonal HA antibody. Membranes were also probed for β-tubulin expression as a loading control. The immunoblots are representative of four separate experiments. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Western Blot, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular detection of NHE6 variants in recycling endosomes in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were incubated with the recycling endosomal marker Alexa Fluor 488–conjugated transferrin (Tf-AF488, 10 μg/ml) for 45 min, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and Tf-AF488 channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with Tf-AF488 as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 6479.8, p value = 5.6 × 10−9), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Incubation, Marker, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Subcellular localization of certain CS variants in the endoplasmic reticulum in transfected AP-1 cells. A, AP-1 cells were transiently transfected with mCherry fluorescent protein-tagged NHE6 (NHE6ChFP) WT or CS-linked variants. Forty eight hours post-transfection, cells were immunostained for endogenous CANX, fixed in 4% paraformaldehyde, mounted onto glass slides, and examined by confocal microscopy. Images show each channel individually, with merged images of the NHE6ChFP and CANX channels. Scale bars represent 10 μm. B, quantitation of the degree of NHE6 overlapping with CANX as determined by calculating the thresholded Mander's coefficient (M1) using ImageJ software and the JACoP plugin. Data are plotted as a box chart, with the central white square indicating the mean, the box representing the S.E., and the error bars showing the S.D. (n = 6–8 cells). Significance from WT was determined by one-way repeated measures ANOVA (F value = 3012.9, p value = 1.7 × 10−10), with a post hoc Dunnett's test, ★★★, p < 0.001.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Transfection, Confocal Microscopy, Quantitation Assay, Software

Journal: The Journal of Biological Chemistry

Article Title: Assorted dysfunctions of endosomal alkali cation/proton exchanger SLC9A6 variants linked to Christianson syndrome

doi: 10.1074/jbc.RA120.012614

Figure Lengend Snippet: Assessment of the functional properties of NHE6 variants. A, biochemical determination of plasma membrane trafficking of NHE6GFP WT or CS-linked variants using a cell-surface biotinylation assay. Cell-surface proteins were labeled with N-hydroxysulfosuccinimidyl–SS–biotin in AP-1 cells expressing the NHE6GFP constructs after 48 h. Total-cell lysates (left panel; protein loading ranged from 10 to 50 μg of protein per sample as indicated below the blot) and biotinylated fractions (right panel; representing 20–100% of plasma membrane proteins extracted per sample) were examined by Western blotting with polyclonal anti-GFP and monoclonal anti-GAPDH antibodies. Representative blots from three experiments are shown. B and C, surface expression and endocytosis of external triple flag tag–labeled NHE6 (3FNHE6HA) constructs in transiently transfected (48 h) AP-1 cells using a cell-based ELISA. Mean intensity fluorescence (M.I.F.) units were determined as a function of the cellular protein concentration and then normalized as percentage (M.I.F. units for WT (100%): 25,100 ± 6,348, n = 4). The surface expression of each construct at time 0 min (before the start of internalization) is charted in B (n = 3–4 experiments). Significance from WT-expressing cells was determined using a one-way repeated measures ANOVA (F value = 463.3, p value = 0.0022), with a post hoc Dunnett's test; *, p < 0.05. Percentage internalization of NHE6 constructs normalized to the zero time point are presented in C and represent the mean ± S.D. (n = 3–4 experiments). The NHE6 variants clustered into two groups: 1) WT, A9S, and R568Q, and 2) L188P, G383D, E547*, and W570*, with variants within each cluster yielding similar statistical values. Significance from WT cells at the 5- and 15-min time points was determined using a one-way ANOVA (F value = 9.43, p value = 4.48 × 10−5), with a post hoc Tukey test; ★, p < 0.05. D, transferrin uptake in HeLa cells transiently transfected (48 h) with GFP or NHE6GFP constructs. The initial uptake (5 min) of Alexa 633–conjugated transferrin (Tf-AF633) was measured in 1 × 104 GFP-positive HeLa cells per experiment by flow cytometry (M.I.F. units for GFP control: 10,204 ± 1554, n = 4). Data were normalized as a percentage and displayed as percent change from GFP control cells. Significance from control cells was determined using a one-way repeated measures ANOVA (F value = 320.7, p value = 3.8 × 10−4), with a post hoc Dunnett's test; ★★, p < 0.001. E, recycling endosomal pH (pHe) was measured in AP-1 cells in the absence or presence of transiently transfected (48 h) NHE6ChFP constructs by fluorescence ratio image analysis of the internalized pH-sensitive probe FITC-conjugated human transferrin (Tf–FITC). Data represent the average endosomal pHe per cell pooled from three separate experiments (8–12 cells per construct/experiment; n = 24–36). Significance was determined by one-way ANOVA (F value = 40.02, p value = 0), with a post-hoc Tukey test; ★★, p < 0.001. Data in B, D, and E are plotted as box charts, with the central white square indicating the mean; the box representing the S.E.; and the error bars showing the S.D. fg, fully-glycosylated; cg, core-glycosylated; d, dimeric; m, monomeric.

Article Snippet: Western blotting For Western blotting analyses, AP-1 and HeLa cells were grown in 10-cm dishes and transiently transfected with 5 μg of plasmid DNA encoding NHE6 GFP or NHE6 HA WT or mutant constructs using Lipofectamine2000 TM (Invitrogen) according to the manufacturer's recommended procedure.

Techniques: Functional Assay, Clinical Proteomics, Membrane, Cell Surface Biotinylation Assay, Labeling, Expressing, Construct, Western Blot, FLAG-tag, Transfection, In-Cell ELISA, Fluorescence, Protein Concentration, Flow Cytometry, Control

Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Effect of epidermal proteases on prosaposin processing. A, Coomassie Brilliant Blue staining of GST-prosaposin. Recombinant GST-prosaposin was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. The band of the recombinant protein can be seen at ∼75 kDa. B, Western blot analysis of prosaposin degradation products. Extracts from differentiated keratinocytes containing prosaposin were incubated with revC14. Western blot analysis were carried out using antibodies to prosaposin (anti-Prosap), saposin A (anti-SapA), sapoins B (anti-SapB), saposin C (anti-SapC), and saposin D (anti-SapD). During incubation with active caspase-14, prosaposin in the extract was degraded into multiple intermediate products. The glycosylated form of saposin A (15 kDa) was detected. Arrowhead, GST-prosaposin; arrow, saposin A. C, co-transfection of pCMV-HA-Prosap, pCMV-HA-revC14, and pCMV-HA-mesotrypsin in growth and differentiated phases. The Western blot was carried out using a specific antibody to each molecule. D, detection of the active form of mesotrypsin in revC14-transfected keratinocytes. Keratinocytes were transfected with pCMV-HA-vector (control), pCMV-HA-revC14, or pCMV-HA-mesotrypsin and further incubated for 24 h in the presence or absence of protease inhibitors. Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis and the presence of mesotrypsin was analyzed by Western blotting using anti-mesotrypsin antibody. Lane 1, pCMV-HA-vector; lane 2, pCMV-HA-revC14; lane 3, pCMV-HA-revC14 + Z-VAD-fmk; lane 4, pCMV-HA-revC14 + leupeptin; lane 5, pCMV-HA-revC14 + Z-VAD-fmk + leupeptin; lane 6, pCMV-HA-mesotrypsin. E, effect of caspase-14 on mesotrypsinogen activation. Enzymatic activity of mesotrypsin was measured using Boc-Gln-Ala-Arg-methylcoumarin amide as a substrate after incubation with caspase-14. To evaluate the direct hydrolytic activity of caspase-14 on this substrate, the same concentration of caspase-14 was incubated without mesotrypsinogen. Amounts of enzymes used in each assay (ng) are listed in parentheses. For comparison, enterokinase was also used. Results are shown as the mean of duplicate experiments. F, Western blot analysis of prosaposin degradation products by mesotrypsin, KLK5, and KLK7. After incubation with each protease, prosaposin degradation products were detected with antibodies to GST, prosaposin, saposin A, saposin B, saposin C, and saposin D. Asterisks indicate the presence of each saposin protein band. Lane 1, prosaposin control; lane 2, prosaposin + mesotrypsin; lane 3, prosaposin + KLK5; lane 4, prosaposin + KLK7.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Staining, Recombinant, SDS Page, Western Blot, Incubation, Cotransfection, Transfection, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Activation Assay, Activity Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Expression and localization of prosaposin and saposin A. A, expression of prosaposin gene transcript in cultured keratinocytes. cDNAs were prepared from cultured keratinocytes at 80% confluence, 100% confluence, 2 days after confluence (120%), 2 days after confluence in the presence of 1.2 mm CaCl2, and 2 days after confluence together with air exposure. The prosaposin mRNA levels were determined using real-time RT-PCR. Data were normalized to the GAPDH gene. B, immunohistochemical localization of mesotrypsin, active caspase-14, prosaposin, and saposin A. Nuclei were counterstained with DAPI. Merged figures of antibody staining images with nuclear staining images are also shown. The dotted line shows the edge of the cornified layer. The broken line shows the epidermal-dermal junction. Scale bars: 50 μm. C, demonstration of interaction by PLA. Interaction between prosaposin, active caspase-14, and mesotypsin was investigated in vivo using the PLA method. Detection was carried out using the following antibody combinations: normal rabbit IgG/anti-Prosap Ab (negative control), anti-mesotrypsin Ab/h14D146, anti-Prosap Ab/anti-mesotrypsin Ab, and anti-Prosap Ab/h14D146. Merged figures of antibody staining images with nuclear staining images are also shown. Scale bars, 50 μm.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Immunohistochemical staining, Staining, In Vivo, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Effects of knockdown of caspase-14 and mesotrypsin on skin equivalent models. A, immunohistochemical analysis of the expression of caspase-14 and mesotrypsin. After treatment of keratinocytes with nonspecific control siRNA, caspase-14 siRNA, mesotrypsin siRNA, or the combination of both, keratinocytes were seeded on dermal components and skin equivalent models were constructed. Thin sections were stained with caspase-14 mAb and anti-mesotrypsin Ab. HE, hematoxylin and eosin stain. B, down-regulation of saposin A in the skin equivalent models after knockdown of caspase-14 and mesotrypsin. Sections from skin equivalent models treated with appropriate siRNAs were stained with anti-Prosap Ab and anti-SapA Ab. Merged figures of antibody staining images with nuclear staining images are also shown. Scale bars, 50 μm.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Immunohistochemical staining, Expressing, Construct, Staining, H&E Stain

Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Prosaposin and saposin A deficiency affects lipid content in the cornified layer. A, immunohistochemical analysis of filaggrin, active caspase-14, and mesotrypsin expression in wild-type and prosaposin-deficient mice. Merged figures of antibody staining images with nuclear staining images are also shown. Scale bars, 50 μm. B, Oil Red staining of skin of prosaposin-deficient and saposin A-deficient mice. Two skin examples each are shown for wild-type mice, prosaposin-deficient mice, and saposin A-deficient mice. Scale bars, 50 μm.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Hsc70 copurifies with cyclin D1. (A) Lysates were fractionated by gel filtration chromatography, and 5% of each fraction was resolved by SDS-PAGE; elution of cyclin D1 (top panel) and CDK4 (bottom panel) was visualized by immunoblot analysis. The positions of eluting molecular weight standards are indicated at the top. (B) Fractions corresponding to lanes 4 to 8 in panel A were immunoprecipitated (IP) with a monoclonal antibody specific for cyclin D1 and either blotted for associated CDK4 (top) or assayed for their ability to phosphorylate recombinant GST-Rb (bottom). (C) Detection of cyclin D1 and cyclin D1-associated proteins by silver stain. Lane 1 contains molecular weight markers (sizes shown in kilodaltons), lane 2 contains proteins that nonspecifically bind to M2 beads from control NIH 3T3 lysates, and lane 3 contains cyclin D1 complexes isolated from FlagD1-T286A-3T3 lysates (an essentially identical pattern was recovered from Flag-D1-3T3 cells; data not shown). Proteins were eluted with excess Flag peptide. Positions of Flag-D1, CDK4, and p21Cip1 are indicated to the right, as is the position of Hsc70, along with peptides identified by mass spectrometry.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Filtration, Chromatography, SDS Page, Western Blot, Molecular Weight, Immunoprecipitation, Recombinant, Silver Staining, Control, Isolation, Mass Spectrometry

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Mitogen-dependent regulation of cyclin D1-Hsc70 binding. (A) Wild-type NIH 3T3 cells were used as an asynchronous culture (asyn) or synchronized by serum deprivation for 36 h and stimulated to reenter the cell cycle by addition of 10% fetal calf serum. Cell lysates were prepared at the indicated intervals (bottom) and immunoprecipitated (IP) with normal rabbit serum (NRS) or a cyclin D1-specific monoclonal antibody (D1). Precipitated proteins were then subjected to immunoblot analysis with antibodies specific for Hsc70 (top) or cyclin D1 (bottom). (B) D1-3T3 cells were synchronized as in panel A. Cell lysates were prepared at the indicated intervals (bottom) and precipitated with normal rabbit serum (NRS) or the M2 monoclonal antibody and subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel) or cyclin D1 (bottom panel). (C) Cell lysates prepared as for panel A were subjected to direct Western analysis with an Hsc70-specific antibody. (D) Whole-cell lysates prepared from asynchronous D1-3T3 cells or D1-3T3 cells cultured in medium containing 0.1% fetal calf serum for the indicated intervals were precipitated with normal rabbit antiserum (NRS) or with the M2 monoclonal antibody. Precipitates were immunoblotted with either Hsc70 (top panel) or cyclin D1 (lower panel) antibodies. The arrow indicates the point at which cells were placed in medium containing 0.1% serum.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Binding Assay, Immunoprecipitation, Western Blot, Cell Culture

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Hsc70 decreases cyclin D1 proteolysis. (A) NIH 3T3 cells were cotransfected with (lane 2) or without (lane 1) a vector encoding Hsc70 along with vectors encoding either cyclin D1 and CDK4 (top and bottom panels), cyclin D1-T286A and CDK4 (second panel), or Myc-tagged cyclin E and CDK2 (bottom panel). Levels of the indicated proteins were determined by direct Western blot analysis of total cell lysates prepared from cells transfected with the indicated vectors. (B) Whole-cell extracts were prepared from NIH 3T3 cells cotransfected with either Flag-D1 and CDK4 (lanes 1 and 2) or Flag-D1, CDK4, and Hsc70 and precipitated with either normal rabbit serum (lane 1) or the M2 monoclonal antibody (lanes 2 and 3). Cyclin D1 and coprecipitating CDK4 were detected by immunoblot analysis with antigen-specific antibodies. (C) NIH 3T3 cells were cotransfected with a plasmid encoding cyclin D1 with (lane 2) or without (lane 1) a vector encoding Hsc70K71E. Levels of cyclin D1 were monitored by immunoblot analysis. (D) p21/p27−/− MEFs were transfected with a plasmid encoding cyclin D1 without (lane 1) or with (lane 2) a plasmid encoding Hsc70. Levels of cyclin D1 were monitored by immunoblot analysis. (E and F) NIH 3T3 cells transfected with vectors encoding either Flag-D1 and CDK4 or Flag-D1, CDK4, and Hsc70 were treated with 50 μg of cycloheximide per ml, and lysates prepared from these cells at the intervals indicated at the bottom of each panel were subjected to immunoblot analysis for cyclin D1. The percentage of cyclin D1 remaining at each time point is indicated at the bottom of each lane.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Plasmid Preparation, Western Blot, Transfection

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Cyclin D1 and Hsc70 are components of multiple complexes. (A) Affinity-purified cyclin D1 complexes were resolved by gel filtration. Ten percent of each fraction was subjected to immunoblot analysis with antibodies specific for Hsc70 (top panel), cyclin D1 (second panel), CDK4 (bottom panel), or p21Cip1 (bottom panel). (B and C) The fraction corresponding to lane 5 in panel A was precipitated with a p21-specific antiserum (B) or an Hsc70 monoclonal antibody (C). Associated proteins were monitored by immunoblot analysis with antibodies specific for the proteins indicated to the left of each panel. (D) The fraction corresponding to lane 5 of part A (158 kDa) was subjected to two rounds of immunodepletion with either normal rabbit antiserum (mock) or the Hsc70-specific monoclonal antibody. Codepletion of cyclin D1 (top panel), CDK4 (middle panel), and p21Cip1 (lower panel) was monitored by immunoblot analysis with the respective antibodies. (E) Asynchronous NIH 3T3 cells were pulse-labeled with [35S]methionine for 15 min (lanes 1 to 6) or pulse-labeled and subsequently chased for 10 min with excess unlabeled methionine for 10 min (lanes 7 to 12). Lysates prepared from these cells were resolved by gel filtration chromatography, and cyclin D1 was precipitated from fractions ranging from 670 kDa to 100 kDa (indicated at the top). Proteins were then analyzed by SDS-PAGE, and cyclin D1 was visualized by autoradiography.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Affinity Purification, Filtration, Western Blot, Immunodepletion, Labeling, Chromatography, SDS Page, Autoradiography

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Reconstitution of a 158-kDa cyclin-CDK complex. (A) Sf9 insect cells were infected with baculoviruses encoding cyclin D1, CDK4, and p21Cip1 with (lanes 1 to 6) or without (lanes 7 to 12) Hsc70. Lysates prepared from these cells were resolved by gel filtration chromatography and subjected to immunoblot analysis for Hsc70 (top panel) or cyclin D1 (bottom panel). The elution positions of molecular weight standards are indicated at the top of each panel. (B) Fractions corresponding to lanes 3 and 4 of part A were precipitated with the cyclin D1 monoclonal antibody. Coprecipitation of Hsc70 was monitored by immunoblotting with the Hsc70 monoclonal antibody. (C) Lysates prepared from Sf9 insect cells infected with baculoviruses encoding either cyclin D1 and CDK4 (lane 1), cyclin D1, CDK4, and Hsc70 (lane 2), cyclin D1, CDK4, and p21Cip1 (lane 3), or all four (lane 4) and metabolically labeled with [35S]methionine were precipitated with the cyclin D1 monoclonal antibody. Positions of Hsc70, cyclin D1, CDK4, and p21Cip1 are indicated to the left of the panel and were verified independently by immunoprecipitation with antigen-specific antibodies (data not shown). Labeled proteins were visualized by autoradiography.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Infection, Filtration, Chromatography, Western Blot, Molecular Weight, Metabolic Labelling, Labeling, Immunoprecipitation, Autoradiography

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Hsc70 maintains cyclin D1-dependent cell cycle progression in the presence of inhibitory levels of p21Cip1. (A) NIH 3T3 cells were transfected with vectors encoding Flag-D1 and CDK4, Flag-D1, CDK4, and p21Cip1, or Flag-D1, CDK4, p21Cip1, and Hsc70; 24 h posttransfection, cells were labeled with bromodeoxyuridine (BrdU) for 20 h. Cells were fixed and stained with monoclonal M2 antibody (red), bromodeoxyuridine (green), and Hoechst dye (blue). (B) Quantitation of panel A, representing three independent experiments; error bars represent standard errors between experiments. (C) Whole-cell lysates prepared from cells transfected as for panel A were resolved by SDS-PAGE, and levels of p21Cip1 were monitored by immunoblot analysis with a p21-specific antibody.

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques: Transfection, Labeling, Staining, Quantitation Assay, SDS Page, Western Blot

Journal:

Article Title: Hsc70 Regulates Accumulation of Cyclin D1 and Cyclin D1-Dependent Protein Kinase

doi: 10.1128/MCB.23.5.1764-1774.2003

Figure Lengend Snippet: Model depicting the participation of chaperones in the regulation of cyclin D1/CDK4 complex maturation (details in text).

Article Snippet: For detection of cyclin D1 complexes, cellular lysates prepared in Tween 20 buffer were resolved on denaturing polyacrylamide gels, transferred to nitrocellulose membranes (Millipore), and blotted with antibodies specific for total cyclin D1 (D1-17-13G), CDK4 (Santa Cruz), Hsc70 (StressGen), or p21 Cip1 (Santa Cruz) or the 9E10 antibody directed against Myc-tagged cyclin E. Sites of antibody binding were visualized with protein A-conjugated horseradish peroxidase (EY Laboratories).

Techniques:

Journal:

Article Title: Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38

doi: 10.1093/emboj/cdf361

Figure Lengend Snippet: Fig. 5. The RalGDS–Ral pathway mediates insulin- and EGF-induced activation of ATF2-dependent gene expression. (A) The effects of insulin and MMS on Ral activity. A14 cells were transiently transfected with 3 µg of pMT2-HA-Ral. At 24 h after transfection, the cells were serum-starved overnight followed by stimulation with either 10 nM insulin (15 min) or 1 mM MMS (2 h). Total cell extracts (750 µg of protein) were incubated with 15 µg of GST–RalBD pre-coupled to glutathione beads to recover GTP-bound Ral. Beads were washed extensively, and collected Ral was detected by immunoblotting with HA antibody. (B) Insulin- and EGF-induced activation of ATF2-dependent transcription is inhibited by RasN17 and RalN28. A14 cells were transiently transfected with 2 µg of either the cJun–ATF2-dependent luciferase reporter 5×jun2-tata or the tata-luciferase control, in the presence or absence of 2 µg of expression vectors for RasN17 and RalN28, or an empty control vector. At 20 h after transfection, the cells were stimulated for 6 h with 10 nM insulin or 1 mM MMS. Depicted is the relative luciferase activity (RLU) ± SD. (C) Dominant-negative Ral inhibits insulin-induced p38 phosphorylation. A14 cells were transiently transfected with 0.5 µg of pMT2-HA-p38 in the presence or absence of 1.5 µg of pMT2-HA-RalN28, or an empty expression vector as described in Figure 2A. Subsequently, the cells were serum-starved and treated with either 10 nM insulin or 500 mM NaCl (O.S.). Total cell extracts were prepared after 15 min, and analyzed by SDS–PAGE/immunoblotting. For better comparison, a relatively short exposure of osmotic shock-induced HA-phospho-p38 is shown. (D) Activation of Ral by RlfCAAX induces p38 phosphorylation. A14 cells were transfected with 0.5 µg pMT2-HA-p38 in the presence or absence of 0.125 µg of HA-RlfCAAX, or an empty vector (–) as described above. At 24 h after transfection, the cells were serum-starved and, after an additional 24 h, total cell lysates were prepared and analyzed by SDS–PAGE and immunoblotting. (E) Activation of Ral by RlfCAAX induces p38 and JNK kinase activity. A14 cells were transfected with 0.5 µg of expression vectors encoding HA-tagged p38, JNK or ERK, respectively, in the presence or absence of 0.125 µg of HA-RlfCAAX expression vector, or an empty vector (–) as described above. At 24 h after transfection, the cells were serum-starved and, after an additional 24 h, total cell lysates were prepared. Lysates were immunoprecipitated with an HA antibody, after which HA-associated ATF2 Thr71 kinase activity was measured using GST–ATF2 as substrate (see Materials and methods). (F) Activation of Ral by RlfCAAX induces ATF2 Thr69 + 71 phosphorylation. A14 and JNK–/– cells were left untreated (–) or transfected with 0.5 µg of pMT2-HA-ATF2 in the presence or absence of 0.125 µg of RlfCAAX expression vector. Fugene reagent was used in order to obtain high levels of transfection efficiency (>40%). At 24 h after transfection, cells were serum-starved overnight, and incubated for a further 24 h in the presence or absence of 10 µM U0126 prior to preparation of cell lysates and analysis by SDS–PAGE and immunoblotting. Note that HA-ATF2 and HA-RlfCAAX (detected by the HA antibody) have nearly the same molecular weight. (G) RlfCAAX enhances transactivation by ATF2 via ATF2 Thr69 and Thr71. A14 cells were transiently transfected with 2 µg of 5×GAL4-E4-luciferase reporter plasmid together with 2 µg of pRSV-GAL4-ATF2 expression vectors containing full-length (wt) ATF2, or the corresponding domain in which Thr69 (T69A), Thr71 (T71A) or both (T69/71A) are replaced by alanine. In addition to these GAL4 fusion constructs, 3 µg of pMT2-RlfCAAX, or an empty expression vector was co-transfected. At 40 h after transfection, cells were harvested and analyzed for luciferase activity. The fold activation depicted represents the ratio between luciferase activity in the presence and absence of RlfCAAX. Values represent the mean ± SD.

Article Snippet: The other antibodies used were: phospho-specific ATF2 Thr69 + 71, ATF2 Thr71, p38 Thr180/Tyr182, ERK hr202/Tyr204 (Cell Signaling Technology), p38 (N-20), ATF-2 (C-19), cJun (H-79), ERK (K-23), donkey anti-goat IgG–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology), monoclonal anti-HA antibody 16B12 (BabCO), anti-GST (Amersham Pharmacia Biotech), phospho-specific JNK Thr183/Tyr185, goat anti-rabbit and goat anti-mouse IgG–HRP conjugate (Promega).

Techniques: Activation Assay, Expressing, Activity Assay, Transfection, Incubation, Western Blot, Luciferase, Plasmid Preparation, Dominant Negative Mutation, SDS Page, Immunoprecipitation, Molecular Weight, Construct

Journal:

Article Title: Growth factors can activate ATF2 via a two-step mechanism: phosphorylation of Thr71 through the Ras-MEK-ERK pathway and of Thr69 through RalGDS-Src-p38

doi: 10.1093/emboj/cdf361

Figure Lengend Snippet: Fig. 4. Insulin- and EGF-induced ATF2 Thr71 mono-phosphorylation is mediated by ERK. (A) The main insulin-induced ATF2 N-terminal kinase activity co-purifies with ERK1/2 after MonoQ anion-exchange chromatography. Total cell lysates from A14 cells treated for 15 min with either 10 nM insulin or 500 mM NaCl (O.S.) were separated on a MonoQ column using a linear gradient of NaCl (dotted line). Fractions were analyzed for in vitro ATF2 kinase activity (filled circles) as described in Materials and methods, and for the presence of JNK, ERK1/2 and p38 by SDS–PAGE and immunoblotting. (B) Insulin induces ERK-associated ATF2 N-terminal kinase activity. Serum-starved A14 cells were either untreated or treated for 15 min with 10 nM insulin, with or without 15 min pre-treatment with 20 µM PD98059, as indicated. Total cell lysates were immunoprecipitated with antibodies that recognize both ERK1 and ERK2, and subsequently assayed for ATF2-kinase activity. (C) Insulin-induced ERK and p38 activities differ in their ATF2 Thr69 and Thr71 kinase activities. Partially purified ERK and p38 preparations from insulin-stimulated A14 cell extracts (MonoQ fractions 12 and 17, respectively) were analyzed for in vitro kinase activity, using either wild-type (wt) or mutant GST–ATF2 fusion proteins in which Thr69 (T69A), Thr71 (T71A) or Thr69 + 71 (T69/71A) were replaced by alanine. The phosphorylation state subsequently was monitored by SDS–PAGE followed by autoradiography and immunoblotting using phospho-specific antibodies followed by enhanced chemiluminescence (ECL). (D) Quantification of 32P incorporation into GST–ATF2 by MonoQ fractions 12 and 17 in in vitro kinase assays as described in (C). The relative activity (mean ± SD) shown represents the 32P incorporation in the various mutant GST–ATF2 substrates relative to that in the wild-type GST–ATF2 protein (set at 100% for both fractions 12 and 17). (E) Recombinant active ERK only phosphorylates ATF2 Thr71 efficiently. Recombinant ERK (10 U; Calbiochem) was compared with MonoQ fraction 13 and total cell lysate from insulin-treated A14 cells for its ATF2 kinase potential using GST–ATF2 as a substrate. The phosphorylation state of GST–ATF2 Thr69 + 71 and GST–ATF2 Thr71 subsequently was monitored by SDS–PAGE/immunoblotting using phospho-specific antibodies. Equal loading of the GST–ATF2 substrate was verified by reprobing the filters with GST antibodies.

Article Snippet: The other antibodies used were: phospho-specific ATF2 Thr69 + 71, ATF2 Thr71, p38 Thr180/Tyr182, ERK hr202/Tyr204 (Cell Signaling Technology), p38 (N-20), ATF-2 (C-19), cJun (H-79), ERK (K-23), donkey anti-goat IgG–horseradish peroxidase (HRP) conjugate (Santa Cruz Biotechnology), monoclonal anti-HA antibody 16B12 (BabCO), anti-GST (Amersham Pharmacia Biotech), phospho-specific JNK Thr183/Tyr185, goat anti-rabbit and goat anti-mouse IgG–HRP conjugate (Promega).

Techniques: Activity Assay, Chromatography, In Vitro, SDS Page, Western Blot, Immunoprecipitation, Purification, Mutagenesis, Autoradiography, Recombinant

Journal: Molecular and Cellular Biology

Article Title: Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

doi: 10.1128/mcb.02279-06

Figure Lengend Snippet: FIG. 2. Perturbation of SIRT1 activity affects secretion of HMW forms of adiponectin. 3T3-L1 adipocytes (4 days) were exposed to either resveratrol (50 M) or nicotinamide (5 mM) in standard DMEM containing 10% FBS for 2 days. The cells were then cultured in fresh DMEM overnight, at which time the medium and total cell layer were harvested for Western blot analysis of intra- and extracellular proteins on reducing (A) and nonreducing (B) SDS-PAGE as outlined in Materials and Methods.

Article Snippet: The antibodies employed in the analysis were as follows: mouse polyclonal antiadiponectin antibody (Affinity BioReagents, Golden, CO), polyclonal anti-SIRT1 antibody (Upstate), anti-PPAR and anti-C/EBP (Santa Cruz Biotechnology), anti-Ero1 polyclonal antibody (Abnova Co., Taiwan, China), and polyclonal anti-aP2 serum (David Benlohr, University of Minnesota).

Techniques: Activity Assay, Cell Culture, Western Blot, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

doi: 10.1128/mcb.02279-06

Figure Lengend Snippet: FIG. 4. Inhibition of SIRT1 expression enhances secretion of HMW adiponectin. 3T3-L1 preadipocytes expressing a control vector or SIRT1 siRNA were differentiated for the indicated number of days, and medium (extracellular) as well as the total cell layer (intracellular) was harvested for Western blot analysis of proteins on reducing (A) or nonreducing (B) SDS-PAGE employing antibodies to the following proteins: SIRT1, PPAR, C/EBP, adiponectin, adipsin, and aP2/FABP4.

Article Snippet: The antibodies employed in the analysis were as follows: mouse polyclonal antiadiponectin antibody (Affinity BioReagents, Golden, CO), polyclonal anti-SIRT1 antibody (Upstate), anti-PPAR and anti-C/EBP (Santa Cruz Biotechnology), anti-Ero1 polyclonal antibody (Abnova Co., Taiwan, China), and polyclonal anti-aP2 serum (David Benlohr, University of Minnesota).

Techniques: Inhibition, Expressing, Control, Plasmid Preparation, Western Blot, SDS Page

Journal: Molecular and Cellular Biology

Article Title: Adiponectin Secretion Is Regulated by SIRT1 and the Endoplasmic Reticulum Oxidoreductase Ero1-Lα

doi: 10.1128/mcb.02279-06

Figure Lengend Snippet: FIG. 6. SIRT1 regulates expression of the ER oxidoreductase Ero1-L. (A) 3T3-L1 preadipocytes were differentiated for the indicated days, and total cell RNA was extracted for RT-PCR analysis of mRNAs corresponding to PPAR, C/EBP, adiponectin, Ero1-L, FABP4/aP2, and GAPDH. (B and C) 3T3-L1 preadipocytes expressing a control vector (-) or a SIRT1 siRNA () were differentiated for 8 days for RT-PCR analysis of mRNAs (B) or for the indicated days for Western blot analysis of intracellular proteins (C). (D) Control and SIRT1 siRNA-expressing 3T3-L1 preadipocytes were differentiated in the absence (Con) or presence of either a PPAR antagonist (Antag; 10 M T0070907) or PPAR agonist (Trog; 5 M troglitazone), and the medium (extracellular) and total cell layer (intracellular) were harvested for Western blot analysis of the indicated proteins.

Article Snippet: The antibodies employed in the analysis were as follows: mouse polyclonal antiadiponectin antibody (Affinity BioReagents, Golden, CO), polyclonal anti-SIRT1 antibody (Upstate), anti-PPAR and anti-C/EBP (Santa Cruz Biotechnology), anti-Ero1 polyclonal antibody (Abnova Co., Taiwan, China), and polyclonal anti-aP2 serum (David Benlohr, University of Minnesota).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Plasmid Preparation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Suppresses Osteopontin-mediated Breast Cancer

doi: 10.1074/jbc.m604403200

Figure Lengend Snippet: FIGURE 1. Expression studies in Rama 37, C9, and TRex R37 subclones. A and B (upper panels), BRCA1 protein expression by immunoblot of wild type (WT.BRCA1), mutant (Mut.BRCA1), and truncated (Trun- .BRCA1) BRCA1 in Rama 37 cells (A) or in the stably transfected C9-Met-DNA Rama 37 subclones versus control parental Rama 37 cells (B) (see “Materials and Methods”). C (upper panel), OPN protein levels by immunoblot in WT.BRCA1 R37, Mut.BRCA1 R37, and Trun.BRCA1 R37 cells versus parental Rama 37 control. Lower panels in A–C show the same blots reprobed for -actin. Positions of authentic BRCA1 (220 kDa), Trun.BRCA1 (50 kDa), -actin (47 kDa), and OPN (75 kDa) are shown in A–C. D and E, expression in C9 subclones. D, OPN mRNA levels in Rama 37 (lane 1), C9 (lane 2), WT.BRCA1 C9 (lane 3), Mut.BRCA1 C9 (lane 4), Trunc.BRCA1 C9 (lane 5), and WT.BRCA1 C9 cells transiently transfected with expression vector for Mut.BRCA1 (lane 6). GAPDH mRNA was assessed as control (lower panel). Major hybridizing bands are shown in kilobases (kb). E, OPN protein levels in the same cell lines as D in identical positions, and positions of authentic OPN and -actin proteins are shown. F, time course (upper panel) and dose response (lower panel) of tetracycline-induced OPN expression in OPN-TRex R37 cells (see “Materials and Methods”). OPN induction was maximal with 1.2 g/ml tetracycline (lower panel). This dose was used in time course studies to show maximal induction at 48 h (upper panel). G and H, OPN expression in TRex R37 sublines. G, OPN mRNA levels in OPN-TRex R37 (lanes 1 and 2), OPN-TRex/WT.BRCA1 R37 (lane 3), OPN-TRex/Mut.BRCA1 R37 (lane 4), OPN-TRex/Trunc.BRCA1 R37 cells (lane 5), and OPN-Trex/WT.BRCA1 cells transiently transfected with an expression vector for Mut.BRCA1 (lane 6) either with () or without () 1.2 g/ml tetracycline, as indicated. GAPDH mRNA was assessed as control (lower panel). Major hybridizing band sizes are shown in kilobases (kb). H, OPN protein levels in the same cell lines and in identical conditions and positions of authentic OPN and -actin proteins are shown. I, endogenous BRCA1 (220 kDa) and OPN (75 kDa) protein levels in Rama 37 cells (R37) or in Rama 37 cells transiently transfected with BRCA1 (BRCA1 siRNA). Panels below blots for OPN in E, F, H, and I show the same blots reprobed for -actin.

Article Snippet: The primary antibodies were as follows: BRCA1 (sc-7867, rabbit polyclonal; Santa Cruz Biotechnology), c-Jun (sc-1694, rabbit polyclonal; Santa Cruz Biotechnology), PEA3 (sc-113, mouse monoclonal; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Mutagenesis, Stable Transfection, Transfection, Control, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Suppresses Osteopontin-mediated Breast Cancer

doi: 10.1074/jbc.m604403200

Figure Lengend Snippet: FIGURE 2. Effects of BRCA1 variants on OPN promoter transactivation. A, WT.BRCA1 concentration-de- pendent effects upon ER/E2-mediated promoter transactivation. Parental Rama 37 cells were cotransfected with the rat OPN promoter luciferase-reporter construct (OPN-Luc), previously determined optimal stimulatory concentrations of expression vector for ER (75 ng) and E2 (108 M) (ER/E2) (see “Materials and Methods”) and 12.5–150 ng of WT.BRCA1 in expression vector DNA. Control experiments used an OPN promoter reporter construct alone. 75 ng of WT.BRCA1 was selected as optimal for subsequent studies. B, BRCA1 protein expres- sionbyimmunoblotintransientlytransfectedRama37cells.Rama37cellswerecotransfectedwiththeratOPN promoter luciferase-reporter construct (lane 1) and 12.5, 37.5, 75, and 125 ng of WT.BRCA1 in expression vector DNA (lanes 2–5), respectively. The resultant cell extracts were analyzed for BRCA1 and -actin by Western blotting as described in Fig. 1, A and B. The positions of authentic BRCA1 and -actin proteins are shown. C, ER/E2-mediated OPN promoter transactivation. Parental Rama 37 cells were cotransfected with the rat OPN promoter luciferase-reporter construct (OPN-Luc), ER/E2, and 75 ng/reaction WT.BRCA1, Mut.BRCA1, or Trun. BRCA1 in expression vectors. D, c-Jun/PEA3-mediated OPN promoter transactivation. Parental Rama 37 cells were cotransfected with OPN-Luc alone (control) or with previously determined optimal concentrations of 200 ng of expression vectors for c-Jun and/or PEA3 alone or in combination together with 75 ng/reaction WT.BRCA1, Mut.BRCA1, or Trun.BRCA1 in expression vectors. Control transfection experiments were per- formed using the pGL-3-Luc (empty luciferase vector), OPN-Luc ER, and OPN-Luc E2. Resulting luciferase values were not significantly different from transfection of OPN-Luc alone (data not shown). A, C, and D, output of the OPN promoter was assessed as firefly luciferase activity at 48 h (see “Materials and Methods”). Values werenormalizedagainstconstitutivelyactivesimianvirus40-drivenRenillaluciferaseandarepresentedasfold induction in excess of transfection of the OPN reporter-luciferase constructs alone (control). Data columns represent the means of three experiments, and error bars represent mean S.D.

Article Snippet: The primary antibodies were as follows: BRCA1 (sc-7867, rabbit polyclonal; Santa Cruz Biotechnology), c-Jun (sc-1694, rabbit polyclonal; Santa Cruz Biotechnology), PEA3 (sc-113, mouse monoclonal; Santa Cruz Biotechnology).

Techniques: Concentration Assay, Luciferase, Construct, Expressing, Plasmid Preparation, Control, Western Blot, Transfection, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Suppresses Osteopontin-mediated Breast Cancer

doi: 10.1074/jbc.m604403200

Figure Lengend Snippet: FIGURE 3. Coimmunoprecipitation of WT.BRCA1 or Mut.BRCA1 with OPN transcription factors. A, WT.BRCA1. 35S-Labeled and nonradioactive pro- teins were produced in cell-free, protein-synthesizing reticulocyte lysates. Coimmunoprecipitation of lysates containing expression vectors for 35S-la- beled WT.BRCA1 and nonradioactive ER (lane 1), nonradioactive c-Jun (lane 3), and nonradioactive PEA3 (lane 5). Lysates programmed to produce only 35S-labeled WT.BRCA1 have been included in lanes 2, 4, and 6. Immunoprecipi- tation was by mAb to ER (lanes 1 and 2), c-Jun (lanes 3 and 4), and PEA3 (lanes 5 and 6), and precipitates were run on polyacrylamide gels. Protein-protein bind- ing of WT.BRAC1 to OPN transcription factors is shown. B, Mut.BRCA1. 35S-La- beled and nonradioactive WT- and Mut.BRCA1 proteins were produced as out- linedabove.Coimmunoprecipitationoflysatescontainingexpressionvectorsfor 35S-labeled WT.BRCA1 and nonradioactive Mut.BRCA1-HA (lane 1), for 35S-la- beled WT.BRCA1 and nonradioactive Tip 60-HA (lane 2), for 35S-labeled WT.BRCA1 only (lane 3), for 35S-labeled WT.BRCA1, and unprogrammed lysates-HA (lane 4) are shown. Immunoprecipitation was by a polyclonal anti- body to HA (sc-805; Santa Cruz Biotechnology) (lanes 1–4), and the resultant precipitates were run on polyacrylamide gels. The position of 35S-labeled WT.BRCA1 protein is indicated (arrow), and its molecular weight is shown. C, IP- Westernblot.DetectionofBRCA1andPEA3proteinsbytheirrespectiveantibod- iesinimmunoblotsofstablytransfectedRama37celllineswitheitherexpression vector for BRCA1 (lane 1) or for PEA3 (lane 2) is shown. In lanes 3 and 4, the cell supernatantscontainingPEA3andBRCA1(lane3)orthesupernatantcontaining PEA3 alone (lane 4) were immunoprecipitated with antibody to BRCA1; the immunoprecipitatesweresubjectedtoPAGE,andtheresultantgelsweresubject to Western blotting for PEA3. The positions of authentic BRCA1 (220 kDa) and PEA3 (75 kDa) are shown by arrows. D, coimmunoprecipitation of BRCA1 with c-Jun or PEA3 in Rama 37 cells. Total cell lysates were immunoprecipitated with eithercontrolIgGorBRCA1antibody.Theimmunoprecipitateswereresolvedby SDS-PAGE and analyzed by Western blotting using anti-BRCA1or anti-c-Jun or anti-PEA3 antibodies as indicated. IP, immunoprecipitation; WB, Western blotting.

Article Snippet: The primary antibodies were as follows: BRCA1 (sc-7867, rabbit polyclonal; Santa Cruz Biotechnology), c-Jun (sc-1694, rabbit polyclonal; Santa Cruz Biotechnology), PEA3 (sc-113, mouse monoclonal; Santa Cruz Biotechnology).

Techniques: Labeling, Produced, Expressing, Immunoprecipitation, Molecular Weight, Plasmid Preparation, Western Blot, SDS Page

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Suppresses Osteopontin-mediated Breast Cancer

doi: 10.1074/jbc.m604403200

Figure Lengend Snippet: FIGURE 4. Effects of a human mutant BRCA1 (Mut.BRCA1) upon WT.BRCA1 repression of OPN promoter- linked luciferase activity. Rama 37 cells were cotransfected with the OPN promoter luciferase reporter (OPN- Luc), with an expression vector for ER with estradiol, and with the expression vectors for wild type BRCA1 (WT.BRCA1) and/or mutant BRCA1 (Mut.BRCA1) in DNA concentrations (ng/reaction) as shown. Results are shown as mean S.D. and fold induction of luciferase activity of triplicate experiments, as described under “Materials and Methods.”

Article Snippet: The primary antibodies were as follows: BRCA1 (sc-7867, rabbit polyclonal; Santa Cruz Biotechnology), c-Jun (sc-1694, rabbit polyclonal; Santa Cruz Biotechnology), PEA3 (sc-113, mouse monoclonal; Santa Cruz Biotechnology).

Techniques: Mutagenesis, Luciferase, Activity Assay, Expressing, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Suppresses Osteopontin-mediated Breast Cancer

doi: 10.1074/jbc.m604403200

Figure Lengend Snippet: FIGURE 5. Effects of BRCA1 and mutants on properties associated with the malignant state of C9-Met- DNA-transformed R37 cells. A, effects on proliferation. Abbreviations used are as described under “Materials and Methods.” Increase in cell number over 3 days is shown; values are the mean S.D. of triplicate experi- ments. B, effects on anchorage-independent growth. Effect of permanent transfection of expression vectors for BRCA1 and mutants on soft agar colony formation of C9 or on Rama 37 cells is shown (106 cells per dish). Cells were grown for 6 days in DMEM as overlays on 0.8% (w/v) agarose beds (see “Materials and Methods”). Results shown represent the mean colony number S.D. of triplicate experiments. Abbreviations are as follows: C9/Mut.BRCA1, Mut.BRCA1 cDNA in an expression vector transfected C9 cells; C9/Trun.BRCA1, Trunc.BRCA1 cDNA transfected C9 cells; C9/WT.BRCA1, WT.BRCA1 cDNA transfected C9 cells, and C9/Mut./ WT.BRCA1, Mut.BRCA1 cDNA transiently transfected WT.BRCA1 C9 cells (see “Materials and Methods”). C, effects on adhesion to fibronectin. Rama 37 (R37) and C9-Met-DNA-transformed R37 subclones (C9 subclones) werepermanentlytransfectedwithexpressionvectorsforBRCA1ormutantsanddesignatedasinBabove.The WT.BRCA1 C9 cells were also transiently transfected with an expression vector for Mut.BRCA1 to yield WT.BRCA1/Mut.BRCA1 C9 cells (C9/Mut./WT.BRCA1). The cells were grown on 20 g/ml fibronectin-coated multiwell plates saturated with 1% (w/v) BSA. Cells were seeded and allowed to attach for 6 h. The number of adherent cells was measured by the A550 of a colorimetric reaction, as described under “Materials and Meth- ods.” Results shown represent the mean S.D. of experiments in triplicate. D, effects on cell invasion. Rama 37 (R37) and C9-Met-DNA transformed subclones (C9): C9, WT.BRCA1 C9, Mut.BRCA1 C9, and Trunc.BRCA1 C9 as well as WT.BRCA1 C9 cells transiently transfected by an expression vector for Mut.BRCA1 (WT.BRCA1/ Mut.BRCA1 C9) were tested; the abbreviations used are those in B above. Cells were cultured in Boyden cham- bers using fibronectin in the lower chamber as a chemoattractant. Cells that invaded through the dividing membrane were fixed, stained, the stain released with 10% (v/v) acetic acid, and the resultant absorbance was measured at 650 nm (see “Materials and Methods”). Results shown represent the mean S.D. of four experiments.

Article Snippet: The primary antibodies were as follows: BRCA1 (sc-7867, rabbit polyclonal; Santa Cruz Biotechnology), c-Jun (sc-1694, rabbit polyclonal; Santa Cruz Biotechnology), PEA3 (sc-113, mouse monoclonal; Santa Cruz Biotechnology).

Techniques: Transformation Assay, Transfection, Expressing, Plasmid Preparation, Cell Culture, Membrane, Staining

Journal: Journal of Biological Chemistry

Article Title: BRCA1 Suppresses Osteopontin-mediated Breast Cancer

doi: 10.1074/jbc.m604403200

Figure Lengend Snippet: FIGURE 7. Osteopontin immunohistochemistry in familial BRCA1 mutant or sporadic BRCA1 wild type breast cancers. Low (A) and high power (B) views of OPN-positive BRCA1 mutant primary breast cancer. C, high power view of a sporadic BRCA1 wild type, OPN negative primary breast cancer. Magnification: A, 90, bar 50 m; B and C, 225; bar 20 m.

Article Snippet: The primary antibodies were as follows: BRCA1 (sc-7867, rabbit polyclonal; Santa Cruz Biotechnology), c-Jun (sc-1694, rabbit polyclonal; Santa Cruz Biotechnology), PEA3 (sc-113, mouse monoclonal; Santa Cruz Biotechnology).

Techniques: Immunohistochemistry, Mutagenesis

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 1 Expression of TAp63a or DNp63a under the control of the K5 promoter in transgenic mice. We obtained two different mouse lines for each isoform, all of which showed similar levels of expression of TAp63a or DNp63a proteins in the basal layer. (a) DNp63 and TAp63 are both expressed in the basal layer, with DNp63 being predominant. (b) The role of the p63 protein is still controversial,23,24 regulating either the stem cells/transient amplifying (TA) cells11 (1) or their differentiation10 (2) or cell death28 (3). The data herein reported are compatible with the first hypothesis, with distinct roles for DNp63 and TAp63. (c) The 5.2 kb K5 constructs used to express TAp63a or DNp63a in basal keratinocytes. Mouse cDNAs are fused in-frame at the N-terminal end with an HA epitope. The distances in kb are indicated in the figure. (d) Expression of the transgene in cultured primary keratinocytes. Western blots for p63 (left, showing endogenous and transgenes) or HA tag (right, showing only transgenes). TA and DN indicate the protein expression in representative transgenic mice (DNp63a or TAp63a). The two lanes on the right show a marker control for both TAp63a and DNp63a proteins. (e) Immunofluorescence for the transgene (stained using an antibody against the HA tag) shows a nuclear localisation for both the TAp63a and DNp63a proteins in primary keratinocytes cultured from the transgenic mice. Bar ¼ 15 mm. (f) Immunohistochemistry of p63 in epidermis, using anti-HA antibody for transgenic mice and anti-p63 (Ab4 clone) for wt mice, showing overexpression of the transgene (brown colour) in the basal layer of the epidermis only. Bar ¼ 120 mm

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Expressing, Control, Transgenic Assay, Construct, Cell Culture, Western Blot, Marker, Staining, Immunohistochemistry, Over Expression

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 2 Reintroduction of TAp63a or DNp63a in p63/ mice by genetic complementation. Morphology (a–d) and haematoxylin and eosin (H&E) skin biopsy staining (e–l) of newborn mice. Bar ¼ 250 mm (e, g, i, k) or 50 mm (f, h, j, l). (a, e, f) wt newborn mice. (b, g, h) p63/ knockout mice. At a gross morphological level, there is no epidermis, allowing the clear visibility of the dermis. Limbs and skin annexes are also absent. Histologically, only few skin patches are present. (c, i, j) p63/ transgenic mice with reintroduction of DNp63a. There is some reformation of the epidermis, as shown by a detachable upper layer. (d, k, l) p63/ transgenic mice with reintroduction of TAp63a. There is no epidermis at a gross morphology level; histologically, the skin is similar to the p63 null mice

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Staining, Knock-Out, Transgenic Assay

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 3 Expression of epidermal differentiation proteins by confocal immunostaining in p63/ mice before and after reintroduction of TAp63a or DNp63a. Colour code and markers of the basal (K5, K14) layers are indicated. Bar ¼ 50 mm. (a) Staining for K14, p63 and Dapi. K14 was visible in very rare patches of the epidermis in the p63/ mice. The reintroduction of DNp63a into the p63/ background allows the reconstruction of the basal layer in several skin areas, with expression of K14. White stars indicate autofluorescent blood cells, also visible in unstained slides. (b) Staining for K5, p63 and Dapi. Like K14, K5 is also re-expressed in p63/;DN transgenic mice. K5 (like K14) was already visible in very few sections of the p63/ mice, as shown. White stars indicate autofluorescent blood cells, also visible in unstained slides. Skin biopsies were taken from newborn mice. (c, d) Colour code and markers of the upper (K1, loricrin) layers are indicated. (c) Staining for K1, p63 and Dapi. K1 is detected in very rare areas of p63/, p63/;DN, p63/;TA complemented mice. (d) Staining for loricrin, p63 and Dapi. Loricrin is detected in very rare areas of upper epidermal layers in p63/, p63/;DN, p63/;TA complemented mice. White stars indicate autofluorescence of blood cells. All bars ¼ 50 mm

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Expressing, Immunostaining, Staining, Transgenic Assay

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 4 Expression of epidermal differentiation proteins by Western blot in p63/ mice before and after reintroduction of TAp63a and/or DNp63a. (a–d) Expression of cornification proteins in p63/ mice after the reintroduction of TAp63a and/or DNp63a. E19.5 embryos were incubated for 24 h in buffer (1% SDS, 20 mM DTT) to solubilise the outermost epidermal layers; proteins were quantified, electrophoresed and blotted. Lanes: 1, p63/;DN;TA ; 2, p63/;TA ; 3, p63/;DN ; 4, p63/ ; 5, wt. (a) K14 expression. A limited, but definite, K14 expression was already detectable in p63/ mice. The reintroduction of the DNp63a protein (but not TAp63a) was sufficient to significantly increase K14 protein expression. (b) Western blot for filaggrin, already expressed at a low level in p63/ mice, indicating the ability of at least some keratinocytes to differentiate and express markers of the upper layers. As for K14 (Figure 3a), the reintroduction of the DNp63a protein (but not TAp63a) was sufficient to significantly increase the expression of filaggrin. (c) Western blot for loricrin. Results were similar to filaggrin. Proteins from wt mice showed either monomer, owing to the solubilisation of loricrin from the L-granules, or heavily crosslinked proteins (indicated by L-arrow, low molecular weight polymers; and H-arrow, high molecular weight polymers). All transgenic mice showed mainly intermediate oligomers and no monomer (formation of L-granules is abnormal in these mice). Again, p63/ mice (and even more mice with reintroduction of the DNp63a protein, L-arrow) show the expression of loricrin, with a significant degree of crosslinked polymers, suggesting that at least some TG have been expressed and activated. (d) Loading control, actin

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Expressing, Western Blot, Incubation, Molecular Weight, High Molecular Weight, Transgenic Assay, Control

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 5 Ultrastructure of epithelial patches following reintroduction of TAp63a or DNp63a in p63/ mice by genetic complementation showing filaggrin granules (white arrows) and loricrin granules (black arrows). (a) wt mouse; cornified squamous cells (top left) overlie the granular layer where numerous filaggrin granules are associated with keratin fibrils. (b) p63/ knockout mouse; fibroblast-like cells overlie a cell with localised cornification of the cell envelope and enlarged loricrin granules. (c) p63/ transgenic mouse with reintroduction of TAp63a; irregular cornified cell envelops contain enlarged loricrin granules and accumulations of a moderately electron-dense material (white arrowheads). (d) p63/ transgenic mouse with reintroduction of DNp63a; flattened cells predominate but cornified cells, with accumulations of a moderately electron-dense material (white arrowheads), are distributed throughout the superficial layers. All bars ¼ 1 mm

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Knock-Out, Transgenic Assay

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 6 Reintroduction of both TAp63a and DNp63a into the p63/ mice. (a) Newborn p63/;DN;TA show significant degree of re-epithelialisation. (b) Histology (haematoxylin and eosin (H&E)). Areas of re-epithelialisation are evident. Bars ¼ 250 mm (upper panel) or 50 mm (lower panel). (c, d) Confocal immunostaining of skin biopsies of newborn mice. Reintroduction of both DNp63a and TAp63a into p63/ mice allows a greater degree of re-epithelialisation, with expression of differentiated basal (K5, K14) and upper (K1, loricrin) layer markers. White stars indicate autofluorescent blood cells, also visible in unstained slides. Bar ¼ 50 mm

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Immunostaining, Expressing

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 7 Ultrastructure of epithelial patches following reintroduction of TAp63a and DNp63a in p63/ mice by genetic complementation showing filaggrin granules (white arrows) and loricrin granules (black arrows). (a–d) TAp63a and DNp63a genetically complemented into p63/ mice. (a) Partially cornified squamous cells predominate in the superficial layers and numerous keratin fibrils are present in most cells of the epithelium; the detail is reported as (b). (b) (detail of (a)) Keratin filaments are associated with filaggrin granules in the granular layer. (c) Epithelial cells (left) are separated from the underlying connective tissue by a basement membrane (curved arrows); the detail is reported in (d). (d) (detail of (c)). Numerous indistinct hemidesmosomes are also present at the base of the epithelial cells (black arrowheads). All bars ¼ 1 mm

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Membrane

Journal: Cell death and differentiation

Article Title: Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice.

doi: 10.1038/sj.cdd.4401926

Figure Lengend Snippet: Figure 9 DNp63a directly transactivates the K14 promoter. (a) Map of the human K14 promoter region. The grey boxes represent p53-like responsive elements; corresponding bps, sequences and restriction enzymes used to generate deleted constructs are indicated (D1–3). (b) DNp63a transactivates the K14 promoter in a dose-dependent manner, as indicated in the luc assay. Deletion of the third p53-like RE completely abrogates transactivation by DNp63a. The luc assay (shown) was performed in Saos-2 cells, and similar results were also obtained in HEK293 cells (not shown). Three independent experiments were performed, and a representative result is shown (mean7S.D., n ¼ 3). (c) ChIP of p63 protein on the K14 promoter using oligonucleotides for the p53-like binding sites 1–2 or 3. The ChIP was performed using nuclear extracts from HaCat cells (lane 1: marker; lane 2: non-specific antibody (nsp); lane 3: specific antibody anti-p63 (sp); lane 4: input. A representative result of two independent experiments is shown. (d) Western blot of K14 induced by DNp63a upon transient transfection in HEK293 cells. Cells were co-transfected with a plasmid containing GFP in order to monitor transfection efficiency. Western blot of actin was used as loading control. A representative result of two independent experiments is shown

Article Snippet: To extract proteins from E19.5 embryonic skin, embryos were incubated at room temperature (RT) for 24 h in 100 mM Tris-HCl pH 8.5, 1% SDS, 20 mM DTT and 5 mM EDTA; 50 mg of extracted proteins was separated on SDS-PAGE and transferred onto polyvinylidendifluoride membranes; blots were kept in blocking solution for 2 h. Blots were incubated for 2 h with shaking at RT with the following primary antibodies: monoclonal anti-p63 (1 : 200 dilution), polyclonal antiHA (Y-11, Santa Cruz, CA, USA, 1 : 100 dilution), polyclonal anti-K14; polyclonal, anti-loricrin and anti-filaggrin (1 : 300 dilution).

Techniques: Construct, Binding Assay, Marker, Western Blot, Transfection, Plasmid Preparation, Control

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: MyoD occupies the Cdc6 promoter in myoblasts. (A) Schematic of the Cdc6 promoter. Closed ovals (E1 and E2) and squares identify the putative MyoD (E-boxes) and E2F sites within the promoter, respectively. The inverted arrows represent the primers used in the PCR amplification reactions. The transcription start site is depicted with an arrow. (B) Cross-linked chromatin from C2C12 myoblasts or primary mouse myoblasts cultured in GM was immunoprecipitated in parallel with anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E1 and E2 sites in the Cdc6 promoter. A control reaction with the use of normal rabbit IgG (NR IgG) is shown along with input DNA (0.05%), which was amplified by the same set of primers. Black lines indicate that intervening lanes have been spliced out. (C) ChIP experiments were performed in parallel on chromatin from C2C12 myoblasts before and after differentiation using normal rabbit IgG or an antibody specific for MyoD. Precipitated DNA was then analyzed by PCR using the E1 primers, and afterward, the bands were quantified by ImageJ (version 1.36b; National Institutes of Health). Controls for PCR included the use of normal rabbit IgG and the titration of input DNA to ensure all amplifications were within the linear range ( Fig. S1 D ). (D) Values in the histogram represent the ratio of chromatin-bound MyoD to input genomic DNA and are the mean of three independent experiments with standard deviation.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Amplification, Cell Culture, Immunoprecipitation, Control, Titration, Standard Deviation

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: The E1 and E2F sites are functionally important to the activity of the Cdc6 promoter. (A) A schematic representation of three luciferase (Luc) reporter constructs, empty vector (pGL3-basic), and a construct driven by a wild-type (wt) Cdc6 promoter (Cdc6-wt) or a Cdc6 promoter harboring mutations within the E1 site (Cdc6-mut1). (B) C3H10T1/2 cells were separately transfected with empty vector and with Cdc6-wt or Cdc6-mut1, either alone or with increasing amounts of a plasmid encoding MyoD (pCMV-MyoD). Activity is expressed as fold increase in comparison with activity observed with empty vector after correcting for transfection efficiency (see Materials and methods). (C) Transcriptional-activity of the empty vector or the Cdc6-luciferase constructs in C2C12 myoblasts with or without mutations within E1, E2F, or both sites. (B and C) Error bars represent SEM.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Activity Assay, Luciferase, Construct, Plasmid Preparation, Transfection, Comparison

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: MyoD is expressed before Cdc6 after C2C12 myoblasts or primary satellite cells transition from quiescence. (A) C2C12 myoblasts were cultured either in GM or in methionine-free medium (−Met) with 1% serum for 36 h and then restimulated with GM for the indicated times. For Western blot analysis, whole cell extracts were prepared, resolved on SDS-PAGE, and then probed with antibodies specific to MyoD, Cdc6, and GAPDH. MyoD and Cdc6 mRNAs were measured by RT-PCR. GAPDH was used as the internal control. White lines indicate that intervening lanes have been spliced out. (B) Isolated extensor digitorum longus myofibers were cultured in basal medium (0 h) before switching to mitogen-rich medium for the activation of satellite cells. The myofibers were fixed at the indicated times and then coimmunostained for Pax7 (green) and MyoD (red) or Cdc6 (red). Immunostaining for Pax7 identifies the satellite cells . Arrows point to the individual satellite cells. Approximately 200 myofibers were analyzed in this experiment. Bar, 20 µm.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Cell Culture, Western Blot, SDS Page, Reverse Transcription Polymerase Chain Reaction, Control, Isolation, Activation Assay, Immunostaining

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: Knockdown of MyoD dramatically reduces the expression of Cdc6 in growth-stimulated quiescent myoblasts. (A) C2C12 myoblasts were separately transduced with lentiviruses expressing two distinct shRNAs specific for MyoD (shMyoD-64 and -66) and a scramble shRNA (shCntrl). After 42 h, the myoblasts were brought to quiescence and then stimulated to grow for the indicated times in GM. (B and C) After stimulation, the myoblasts were harvested, and the expression of MyoD or Cdc6 was then monitored either by RT-PCR (B) or by Western blotting (C). NS denotes a nonspecific band. EV, empty vector. (D and E) Cross-linked chromatin was isolated from growth-stimulated quiescent myoblasts expressing a scramble shRNA (shCntrl) or an shRNA specific for MyoD (shMyoD-66). Afterward, the protein–DNA fragments were immunoprecipitated in parallel with anti-E2F3a or anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E1 site within the Cdc6 promoter. A control reaction with the use of normal rabbit IgG (NR IgG) is shown along with input DNA, which was amplified by the same set of primers. Black lines indicate that intervening lanes have been spliced out.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Knockdown, Expressing, Transduction, shRNA, Reverse Transcription Polymerase Chain Reaction, Western Blot, Plasmid Preparation, Isolation, Immunoprecipitation, Control, Amplification

Journal: The Journal of Cell Biology

Article Title: Activation of Cdc6 by MyoD is associated with the expansion of quiescent myogenic satellite cells

doi: 10.1083/jcb.200904144

Figure Lengend Snippet: Myf5 can occupy the promoter of Cdc6 in MyoD −/− primary myoblasts and in MyoD-depleted quiescent myoblasts after serum stimulation. (A and B) Cross-linked chromatin prepared from quiescent C2C12 myoblasts expressing shMyoD-66 after stimulation by serum for periods of 6 or 12 h was immunoprecipitated in parallel with anti-Myf5 and normal rabbit IgG (NR IgG). Semiquantitative PCR was then performed with the use of primers surrounding the E-box site E1 within the Cdc6 promoter. Black lines indicate that intervening lanes have been spliced out. (C) Crossed-linked chromatin from cultured MyoD −/− primary myoblasts was immunoprecipitated in parallel with anti-Myf5 or anti-MyoD antibody (Ab) and then analyzed by semiquantitative PCR using primers surrounding the E-box sites (E1 and E2) within the Cdc6 promoter. A control reaction with the use of normal rabbit IgG is shown along with input DNA, which was amplified by the same set of primers.

Article Snippet: Antibodies recognizing MyoD (C-20 and M-318), Myf5 (C-20), E2F3a (C-20), Cdc6 (180.2 and H-304), E2F4 (C-20), E2F1 (KH95), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 6C5), and normal rabbit IgG were obtained from Santa Cruz Biotechnology, Inc. Hybridoma cells producing the anti-Pax7 mouse monoclonal antibody were provided by M.A.

Techniques: Expressing, Immunoprecipitation, Cell Culture, Control, Amplification

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Schematic showing the domain organization of the reference HER2-specific CAR constructs and modifications made to introduce programmed membrane protein (proMP) transmembrane domains (TMDs). Bold, boxed sequence indicates the human CD28 TMD in the reference CD28TM and no cys CARs and designed proMP sequences in the monomeric (proCAR-1), dimeric (proCAR-2), and trimeric (proCAR-3) receptors. ( b ) BW5147 murine thymoma cells stably expressing proCARs and a destabilized GFP NF-κB reporter were surface labeled with anti-Myc antibody and analyzed by flow cytometry to assess surface expression levels. ( c ) Live cells from ( b ) were coated with polyclonal anti-IgG to bind CARs through the scFv domain and immunoprecipitated using protein G beads. Products were separated by nonreducing SDS-PAGE and immunoblotted using anti-Myc antibody to visualize surface-expressed CAR proteins. Molecular weight of the unglycosylated CAR polypeptide is 55 kDa. ( d, e ) Cells from ( b ) were co-cultured with HER2+ SKBR3 human breast adenocarcinoma cells for the indicated times and analyzed by flow cytometry for upregulation of activation marker CD69 ( d ) and GFP expression from the NF-κB reporter ( e ). All activation levels are normalized to the 8 hr time point in cells expressing the CD28TM CAR (% CD28TM Max). Bars represent the mean ± SD, and dots show the individual data points for three independent experiments. ( f ) Maximum target killing percentage at 20:1 effector to target ratio from 4 hr 51 Cr release assay. Bars show mean ± SEM with each data point representing an individual experiment (n = 3). p-Values determined from paired t -tests. ( g ) Cytokine production by primary mouse HER2 proCAR T cells following 24 hr co-culture with MC57-HER2 target tumor cells. Bars show mean concentration ± SEM with each data point representing an individual experiment (n = 5). Significance was determined from one-way ANOVA with multiple comparisons. Cytokine production on antigen-negative parental MC57 cells shown separately in .

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Construct, Introduce, Sequencing, Stable Transfection, Expressing, Labeling, Flow Cytometry, Immunoprecipitation, SDS Page, Molecular Weight, Cell Culture, Activation Assay, Marker, Release Assay, Co-Culture Assay, Concentration Assay

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Model of the CD28TM interface generated by mutagenesis of the CD3ζ TMD (PDB: 2HAC). Polar residues of the CD28 dimerization motif (orange) with predicted hydrogen bonds depicted (dotted lines). ( b ) Surface expression and ( c ) SDS-PAGE and immunoblot analysis of HER2 CARs possessing WT CD28TM or CD28TM mutations depicted in ( a ) expressed in the BW5147 cell line. ( d ) Quantitation of target cell killing measured by chromium release assay and cytokine production by primary mouse CD8 + CAR T cells in response to the MC57-HER2 target cell line (n = 4). Experiments performed as in . p-Values determined by paired t -tests. ( e ) Representative immunofluorescent confocal images of CAR-CD28 co-clustering in primary mouse CAR T cells. CAR clustering was induced with anti-Myc primary followed by crosslinking with fluorescent secondary antibody (magenta). Cells were then labeled for CD28 (cyan). Images are Z-projections over 12 m, scale bar represents 3 m. ( f ) Quantitation of CAR-CD28 co-clustering, each dot representing the percentage of CAR clusters in one cell that co-localized with a CD28 cluster. Lines show mean CAR-CD28 co-clustering percentage/per cell ± SEM, n ≥ 30 cells. p-Values determined by unpaired t -tests.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Generated, Mutagenesis, Expressing, SDS Page, Western Blot, Quantitation Assay, Release Assay, Labeling

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Flow cytometry gating strategy to determine the transduction efficiency of primary murine CAR T cells. Lymphocytes selected via morphology, live cells selected as zombie aqua negative, T cells selected as CD3 + CD8 + and mCherry + cells defined as CAR T cells. c-Myc co-expression with mCherry indicates surface CAR expression. ( b ) Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM CARs and ProCARs 1–3, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Transduction, Expressing, Labeling, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM chimeric antigen receptors (CARs) and proCAR-4, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Labeling, Transduction, Expressing, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Fluorescence, Microscopy, Recombinant, Sequencing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Modification, Software

Journal:

Article Title: A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

doi:

Figure Lengend Snippet: Interaction of TSG101 with p53 and MDM2. Constructs expressing human TSG101, p53, or MDM2 proteins (2 μg of DNA for each plasmid) were introduced by transfection into Saos-2 cells, as indicated in Materials and Methods. Protein extracts from transfected cell populations were immunoprecipitated by the antibodies indicated. IP, immunoprecipitation; IB, immunoblotting; α-, anti-. (a and b) Native or HA-tagged TSG101 or p53 proteins in Western blots were detected by anti-p53 monoclonal antibody (a; AB-1; 1:1000; the secondary antibody was HRP-conjugated goat anti-mouse IgG, diluted 1:1000) or by anti-HA antibody labeled with HRP (b; diluted 1:500). (c and d) Western blot detection of immunoprecipitated proteins analyzed with anti-MDM2 antibody.

Article Snippet: Antibodies used for Western blots were rabbit anti-TSG101 (1:200, CLONTECH), anti-p53 (DO-1, 1:1000, Santa Cruz Biotechnology), anti-HA (1:500, HRP-labeled, Roche), and anti-Flag (1:500, M2, Kodak), anti-α-tubulin (1:20000, Neomark), anti-rabbit IgG (1:5000, HRP-labeled, Promega) and anti-mouse IgG (1:10,000, HRP-labeled, Santa Cruz Biotechnology).

Techniques: Construct, Expressing, Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Labeling

Journal:

Article Title: A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

doi:

Figure Lengend Snippet: Effect of expression of TSG101 on the cellular level of p53 and the degradation of MDM2. (a) The indicated constructs overexpressing p53 and HA-TSG101 were introduced into Saos-2 cells by transfection, and p53 and HA-TSG101 levels were analyzed by Western blotting 48 h later. + indicates 25 ng of transfected p53 expression vector DNA or 4 μg of HA-TSG101 expression vector DNA; ++ indicates 8 μg of HA-TSG101 expression vector DNA. The density of p53 bands relative to cellular α-tubulin was determined by scanning of exposed films. (b) The identical constructs overexpressing p53 and HA-TSG101 were introduced into p53−/−/MDM2−/− MEF cells by transfection, and p53 and HA-TSG101 levels were analyzed as in a. (c) Constructs overexpressing the proteins indicated were introduced into Saos-2 cells by transfection and p53 and MDM2 levels were analyzed as in a. + indicates 25 ng of transfected p53 expression vector DNA, 1 μg of MDM2 expression vector DNA, or 4 μg of TSG101 expression vector DNA. (d) Saos-2 cells were transfected by an MDM2-expressing construct in the absence or presence of a TSG101-expressing construct. Cellular protein was extracted after addition of cycloheximide at the indicated times (min) and analyzed by Western blotting as in a, using anti-MDM2 antibody. A GFP expression vector was cotransfected to normalize transfection efficiency; the expressed GFP protein was detected with anti-GFP antibody. (e) Plot of degradation of MDM2 for the experiment shown in d, which was representative of five separate experiments. ○, Absence of TSG101; ■, presence of TSG101.

Article Snippet: Antibodies used for Western blots were rabbit anti-TSG101 (1:200, CLONTECH), anti-p53 (DO-1, 1:1000, Santa Cruz Biotechnology), anti-HA (1:500, HRP-labeled, Roche), and anti-Flag (1:500, M2, Kodak), anti-α-tubulin (1:20000, Neomark), anti-rabbit IgG (1:5000, HRP-labeled, Promega) and anti-mouse IgG (1:10,000, HRP-labeled, Santa Cruz Biotechnology).

Techniques: Expressing, Construct, Transfection, Western Blot, Plasmid Preparation

Journal:

Article Title: A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

doi:

Figure Lengend Snippet: MDM2-dependent proteolysis of TSG101. (a) Cellular TSG101 protein levels detected by Western blotting with protein extracts (40 μg each) from mouse fibroblast NIH 3T3 cells, p53−/− MEFs, and MEFs mutated in both p53 and MDM2. Relative densities of TSG101 protein bands were calculated after normalization to cellular α-tubulin. (b) Cell cultures of NIH 3T3, p53−/− MEFs and p53−/−/MDM2−/− MEFs were pulse-labeled with [35S]methionine for 1 h, and chased for 0, 2, 4, 8, and 12 h. The 35S-labeled TSG101 was immunoprecipitated by anti-TSG101 antibody, resolved by electrophoresis in an SDS gel, and visualized and quantitated by ImageQuant. (c) Protein extracts from SJSA-1, U2OS, and Saos-2 cells were analyzed by Western blotting using antibodies to MDM2, p53, TSG101, and α-tubulin. The ratio MDM2/TSG101 was determined after normalization with cellular α-tubulin. (d) Vectors expressing Flag-tagged TSG101 and MDM2 were introduced into Saos-2 cells by cotransfection. Transfecting amounts of DNA are designated by + for 0.5 μg of Flag-TSG101 expression vector and + to ++++ for 1 to 4 μg of MDM2 expression vector. (e) Combinations of vectors expressing HA-tagged TSG101, MDM2, and/or wild-type (WT) or mutant (MT) p53 protein were introduced by transfection into Saos-2 cells, and protein extracts of transfectants were analyzed by Western blotting using antibody as indicated. Where indicated, transfectants received 1 μg of plasmid DNA expressing HA-TSG101, 1 μg of plasmid DNA expressing MDM2, and p53-expressing constructs as follows: + and ++, 1 μg and 2 μg, respectively. The p53 mutation replaced the Arg at amino acid 175 with His. (f) Saos-2 cell cultures were treated as indicated with proteasome inhibitor MG132 (2 μM) for 24 h, and cellular protein extracts were analyzed by Western blotting with anti-TSG101 antibody. (g) The indicated constructs were introduced into Saos-2 cells by transfection (concentrations designated as in d). Transfected cells were cultured in the absence or presence of MG132 for 24 h, and protein extracts of cells were analyzed by Western blotting with anti-HA and anti-MDM2 antibody. The intensity of HA-TSG101 and MDM2 protein bands is indicated relative to cellular α-tubulin.

Article Snippet: Antibodies used for Western blots were rabbit anti-TSG101 (1:200, CLONTECH), anti-p53 (DO-1, 1:1000, Santa Cruz Biotechnology), anti-HA (1:500, HRP-labeled, Roche), and anti-Flag (1:500, M2, Kodak), anti-α-tubulin (1:20000, Neomark), anti-rabbit IgG (1:5000, HRP-labeled, Promega) and anti-mouse IgG (1:10,000, HRP-labeled, Santa Cruz Biotechnology).

Techniques: Western Blot, Labeling, Immunoprecipitation, Electrophoresis, SDS-Gel, Expressing, Cotransfection, Plasmid Preparation, Mutagenesis, Transfection, Construct, Cell Culture

Journal:

Article Title: A TSG101/MDM2 regulatory loop modulates MDM2 degradation and MDM2/p53 feedback control

doi:

Figure Lengend Snippet: Model showing functional interactions of the TSG101/MDM2 and p53/MDM2 feedback control loops.

Article Snippet: Antibodies used for Western blots were rabbit anti-TSG101 (1:200, CLONTECH), anti-p53 (DO-1, 1:1000, Santa Cruz Biotechnology), anti-HA (1:500, HRP-labeled, Roche), and anti-Flag (1:500, M2, Kodak), anti-α-tubulin (1:20000, Neomark), anti-rabbit IgG (1:5000, HRP-labeled, Promega) and anti-mouse IgG (1:10,000, HRP-labeled, Santa Cruz Biotechnology).

Techniques: Functional Assay