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: Vaccines

Article Title: Anti-Influenza Protective Efficacy of a H6 Virus-Like Particle in Chickens

doi: 10.3390/vaccines8030465

Figure Lengend Snippet: Generation and purification. A schematic diagram of H6 virus-like particle (VLP) construction and expression. ( A ) A schematic diagram of H6 VLP construction and expression. The recombinant plasmid carrying the hemagglutinin (HA) and matrix protein 1 (M1) genes derived from the H6 avian influenza virus (AIV) was transposed onto the bacmid DNA. ( B ) VLPs that assembled HA and M1 proteins were harvested in the rBac-H6M1-infected Sf21 culture supernatant. ( C ) The H6 VLPs were purified through sucrose density gradient centrifugation. ( D ) The hemagglutination activity of purified VLPs was verified with the HA test. ( E ) The expression of the HA and M1 proteins on the VLPs was analyzed using SDS-PAGE and validated by Western blot using the antiserum directed against H6 AIVs.

Article Snippet: The HA content of a given VLP was analyzed by comparing the signals generated from the blots with those of the recombinant H6 HA protein (Sino Biological, #11723-V08H, Beijing, China).

Techniques: Purification, Expressing, Recombinant, Plasmid Preparation, Derivative Assay, Infection, Gradient Centrifugation, Activity Assay, SDS Page, Western Blot

Journal: Vaccines

Article Title: Anti-Influenza Protective Efficacy of a H6 Virus-Like Particle in Chickens

doi: 10.3390/vaccines8030465

Figure Lengend Snippet: Characterization of H6 VLPs. ( A ) The size and surface zeta potential of H6 VLPs and native virions were measured using NanoSight. ( B ) The H6 VLPs and native virions were imaged using TEM under negative staining and immunogold staining using the H6/HA monoclonal antibody. Bar = 100 nm.

Article Snippet: The HA content of a given VLP was analyzed by comparing the signals generated from the blots with those of the recombinant H6 HA protein (Sino Biological, #11723-V08H, Beijing, China).

Techniques: Negative Staining, Staining

Journal: Vaccines

Article Title: Anti-Influenza Protective Efficacy of a H6 Virus-Like Particle in Chickens

doi: 10.3390/vaccines8030465

Figure Lengend Snippet: The immunogenicity of H6 VLPs with ISA 71 VG in a chicken model. ( A ) Specific-pathogen-free (SPF) chickens were prime-boost vaccinated on Days 0 and 21 with PBS, VLPs alone, or VLPs with commercial adjuvant ISA 71 VG, and blood samples were collected. H6N1 AIV-specific IgG titers were analyzed by ELISA on day 21. ( B ) H6N1 AIV-specific IgG titers were analyzed by ELISA on day 56. ( C ) H6N1 AIV-specific HI titers were analyzed by a HI test. Error bars represent the means ± S.D. The horizontal dotted lines mark the detection limit for the antibody titer. Serum IgG titers among groups were compared using one-way ANOVA followed by Dunnett’s multiple-comparison test. The HI titers among vaccination groups and the control group were compared using two-way ANOVA followed by Tukey’s multiple-comparison test, and the differences between the VLP alone and ISA 71 VG-adjuvanted groups are shown (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).

Article Snippet: The HA content of a given VLP was analyzed by comparing the signals generated from the blots with those of the recombinant H6 HA protein (Sino Biological, #11723-V08H, Beijing, China).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: Anti-Influenza Protective Efficacy of a H6 Virus-Like Particle in Chickens

doi: 10.3390/vaccines8030465

Figure Lengend Snippet: The protective efficacy of H6 VLPs in a chicken model. ( A ) The vaccine immunization and virus challenge schedule in chickens. Three weeks after the booster immunization, chickens were intranasally challenged with 10 6 50% egg-infective dose (EID 50 ) H6 AIV. ( B ) Viral RNA in tears at 4 dpc was analyzed by RT-qPCR. Error bars represent the means ± S.D. The horizontal dotted line marks the detection limit for viral copies. Viral copies among groups were compared by one-way ANOVA followed by Tukey’s multiple-comparison test.

Article Snippet: The HA content of a given VLP was analyzed by comparing the signals generated from the blots with those of the recombinant H6 HA protein (Sino Biological, #11723-V08H, Beijing, China).

Techniques: Quantitative RT-PCR

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: EGF induces translocation of EGFR to the Golgi. (a) HeLa cells were transfected with pDsRed-syntaxin 6. Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. EGFR was labeled with the indicated antibodies. The boxed areas are shown in detail in the insets. Insets 2–1 and 2–2 show representative colocalizations of EGFR and syntaxin 6. Scale bar, 10 μm. (b) Cells were serum starved overnight and then treated without or with EGF (50 ng/ml) for 20 min. Endogenous EGFR and syntaxin 6 were labeled with a primary antibodies and secondary fluorescein isothiocyanate (donor, green) and Texas-Red (acceptor; red) antibody. An Fc image was obtained using the Zeiss ZEN software. Scale bar, 20 μm. Quantitation of the FRET intensity is shown in the right. (c) Cell lysate was loaded onto the 0–30% OptiPrep density gradient medium and subjected to ultracentrifugation, and fractions were separated using the Gradient Station. The early endosome, the Golgi and ER markers were used to analyze fractions. S, short expose; L, long expose. (d) HeLa cells were treated with or without EGF (50 ng/ml) for 20 min after starvation overnight. The EGFR levels in the Golgi-enriched fraction (fraction 9) were analyzed using immunoblotting. (e) Cells were serum starved overnight and then treated with EGF (50 ng/ml) for 20 min. One cell was used for z-stack scanning. Representative images were shown. The boxed areas are shown in detail in the insets. Scale bar, 10 μm. (f) Cells were transfected with GalNac T2 for 48 h or direct staining of endogenous marker, GM130. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for indicated time and analyzed using confocal microscope. Scale bar, 20 μm. Quantitation of colocalization of EGFR and endosomal markers is shown in the bottom. (g) HeLa cells were transfected with EGFP-GalNac T2. Cells were exposed to serum-free media overnight following treatment without or with EGF (50 ng/ml) for indicated time. Scale bar, 20 μm. The boxed areas are shown in the insets. Quantitation of colocalization of phospho-EGFR and total EGFR with the GalNac T2 is shown in the bottom. (h) HeLa cells were serum-starved overnight before EGF stimulation for indicated time. Total lysate and the Golgi-enriched fractions were performed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western blot to examine the phospho-1086 of EGFR and total EGFR levels.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Labeling, Software, Quantitation Assay, Western Blot, Staining, Marker, Microscopy, Polyacrylamide Gel Electrophoresis

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for the Golgi translocation of EGFR. (a) Cells were first transfected with syntaxin 6 or control (Ctrl) siRNAs for 24 h and then transfected with GalNac T2 for 48 h. Cells were then maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (b) Cells were transfected with syntaxin 6 or control siRNAs. After 72 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. The EGFR levels in the Golgi-enriched fraction were analyzed using immunoblotting. (c) Cells were transfected with CCD domain of syntaxin 6 or control vector. After 48 h transfection, cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min. Cells were analyzed by confocal microscope. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Results of quantitation of colocalization of EGFR and Golgi marker are shown in the right panel. (d) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and was restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated without or with EGF (50 ng/ml) for 20 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (e) Cells were transfected with syntaxin 6 or control siRNAs. After 24 h transfection, cells were transfected with GalNac T2 for 48 h. Cells were maintained in serum-free media overnight and treated without or with EGF (50 ng/ml) for 20 min and then analyzed by confocal microscopy. Scale bar, 20 μm. The boxed areas are shown in detail in the insets. Quantitation of colocalization of EGFR and endosomal markers is shown in the right. (f) HeLa cells were serum-starved overnight and stimulated without or with EGF (50 ng/ml) for 20 min. Cell lysates were immunoprecipitated with the indicated antibodies and subjected to immunoblot analysis as indicated. (g) In vitro transcribed and translated biotin-labeled syntaxin 6 was incubated with recombinant GST-fused EGFR fragments, pulled down using glutathione-Sepharose beads and visualized with horseradish peroxidase (HRP) conjugated streptavidin. CT, c-terminal domain; IB, immunoblot; KD, kimase domain fragment; TM, transmembrane domain fragment.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Confocal Microscopy, Quantitation Assay, Marker, Western Blot, Plasmid Preparation, Microscopy, shRNA, Immunoprecipitation, In Vitro, Labeling, Incubation, Recombinant

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Microtubules and dynein are required for EGF-induced Golgi transport of EGFR. (a) Serum-starved cells were treated with EGF. Double staining of EGFR and α-tubulin were subjected to confocal microscopy assay. Scale bars, 20 μm. (b) HeLa cells were transfected with GFP-GalNac T2, treated with microtubules or dynein inhibitors and then stimulated with EGF. The Golgi-enriched fractions were purified and subjected to immunoblot analysis with the indicated antibodies. (c) Serum-starved HeLa cells were treated as shown in (b) and then stimulated with EGF and analyzed by a confocal microscope. Scale bars, 20 μm. The boxed areas are shown in detail in the insets. Representative colocalization of EGFR and GalNac T2 is shown in inset 2–1. Quantitation of cells with Golgi-localized EGFR is shown in the lower panel. (d) HeLa cells were transfected with GFP-GalNac T2 expression plasmid and then transfected with control (ctrl) vector or CDK1 and cyclin B plasmids, respectively. Cells were then serum starved overnight, stimulated with EGF and further analyzed under a confocal microscope. Scale bar, 20 μm. Quantitative results are shown in the right. (e) Representative frames of time-lapse confocal microscopic image of cells treated with or without nocodazole. HeLa cells were transfected with EGFP–EGFR (green) and DsRed–syntaxin 6 (red) plasmids. After serum starvation overnight and EGF stimulation, images were collected at 30-s intervals as indicated. Scale bar, 5 μm. (f) Serum-starved HeLa cells were transfected with dynein shRNAs and then stimulated with EGF. Golgi-enriched fractions were purified and subjected to immunoblot analysis with indicated antibodies. DMSO, dimethyl sulfoxide; Noc, nocodazole; PT, paclitaxel; Van, vanadate.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Double Staining, Confocal Microscopy, Transfection, Purification, Western Blot, Microscopy, Quantitation Assay, Expressing, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Syntaxin 6 is required for EGFR nuclear translocation. (a) HeLa cells were transfected with syntaxin 6 or control siRNAs and maintained in a serum-free media overnight and treated with EGF (50 ng/ml) for 30 min. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (b) Cells were transfected with syntaxin 6 or control siRNA and maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (c) Cells were transfected with syntaxin 6 shRNA targeting to the 3′-UTR region or control shRNA. Syntaxin 6 and vector control were restored in cells with knockdown of endogenous syntaxin 6. Cells were maintained in serum-free media overnight and then treated with EGF (50 ng/ml) for 30 min. Cellular fractions were subjected to immunoblotting with the indicated antibodies. (d) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Quantitation of positive cells with nuclear EGFR is shown in the lower panel. Scale bar, 20 μm. (e) HeLa cells were transfected with a control vector and syntaxin 6 CCD and maintained in serum-free media overnight, and then stimulated with EGF. Nuclear and non-nuclear fractions were subjected to immunoblot analysis with the indicated antibodies. DAPI, 4′,6-diamidino-2-phenylindole.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Translocation Assay, Transfection, Quantitation Assay, Western Blot, shRNA, Plasmid Preparation

Journal: Oncogene

Article Title: Syntaxin 6-mediated Golgi translocation plays an important role in nuclear functions of EGFR through microtubule-dependent trafficking

doi: 10.1038/onc.2013.1

Figure Lengend Snippet: Nuclear function of EGFR requires syntaxin 6 and microtubules. (a) After overnight serum starvation, cells were pretreated with the indicated inhibitors for 30-min treatment and then stimulated with EGF for 30 min, followed by chromatin-IP assay. For IgG control, lysate of cells without EGF stimulation was used. (b) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for 30 min, followed by chromatin-IP assy. For IgG control, lysate of cells without EGF stimulation was used. (c) Cells were transfected with siRNAs of syntaxin 6. After 72 h transfection, cells were serum starved overnight and then stimulated with EGF for indicated time. Quantitative reverse transcription–polymerase chain reaction (RT–PCR) was used to analyze the mRNA level. (d) HeLa cells transfected with control siRNAs and siRNAs for syntaxin 6 were transfected with reporter plasmids containing CCND1 promoter. Then, after 24 h transfection, cells were maintained in serum-free media overnight and treated with EGF for indicated time. Total lysates were used for luciferase assay. Error bars were derived from three independent experiments. (e) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After transfection, 4 × 105 cells were seeded in a six-well plate, incubated for 72 h and then counted. (f) HeLa cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 48 h transfection, cells were treated with BrdU (100 μm) for 1 h. Cells were assayed for BrdU incorporation by flow cytometry. (g) BT20 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (h) OVCAR3 cells were transfected with control siRNAs and siRNAs for syntaxin 6. After 24 h transfection, 2 × 105 cells were seeded in a 12-well plate overnight, treated with 0.1, 1 and 10 μm of gefitinib for 72 h and then counted. (i) A schematic model of syntaxin 6- and microtubule-mediated Golgi and nuclear transport of EGFR.

Article Snippet: The syntaxin 6 full-length plasmid was purchased from OriGene (Rockville, MD, USA), which was subcloned into pDsRedC1 (Clontech, Mountain View, CA, USA) for fluorescence staining.

Techniques: Chromatin Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Luciferase, Derivative Assay, Incubation, BrdU Incorporation Assay, Flow Cytometry

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured in low or high density. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without serum. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. Immunoblot estimation of GFP‐hcGAS in nuclear/cytosolic fractions and corresponding flow cytometric analysis of cell cycle of HEK293 cells cultured with or without aphidicolin. Lamin B and α‐tubulin are nuclear and cytosolic markers, respectively. cGAS in nuclear/cytosolic fractions of indicated cell types. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Western Blot, Cell Culture

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Fluorescence images of GFP‐hcGAS, GFP‐hcGASΔcGAMP, GFP‐hcGASΔDNA, and GFP‐hcGASΔOligo in HEK293 cells cultured with or without aphidicolin. Scale bar: 20 μm. B Corresponding quantification of (A). The nuclear cGAS/total cGAS was calculated from 6 different fields with n > 50 cells. C, D A nuclear export signaling (NES) is not sufficient to dislodge chromatin‐bound cGAS from the nucleus. (C) Fluorescence images of GFP‐hcGAS, GFP‐hcGAS‐NLS, and GFP‐hcGAS‐NES in HEK293 cells. Scale bar: 10 μm. (D) Immunoblots of subcellular fractions of GFP‐hCGAS‐, GFP‐hCGAS‐NLS‐, and GFP‐hCGAS‐NES‐expressing HEK293 cells. Data information: Data are presented as means ± SEM. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS: P > 0.05 and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Fluorescence, Cell Culture, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A, B Micronuclei (indicated by arrowhead) in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells before (0 h) or 24 h after γ‐irradiation (IR; 10 Gy). Scale bar: 10 μm (A). (B) The average MNs/cell. Graphs show mean ± SEM ( n = 3 independent experiments) representing six different microscopic fields with over 200 cells. C IFNB1 response in HEK293 cells stimulated with transfected plasmid DNA. Mean ± SEM of n = 3 independent experiments. D Experimental outline for micronucleus generation and cell death after γ‐irradiation. E Micronucleus (indicated by arrowhead) and cGAS staining in WT and cGAS −/− BMDMos exposed to γ‐irradiation (10 Gy). Scale bar: 10 μm. F Average MNs/cell in BMDMos. MN graphs show mean ± SEM ( n = 3 independent experiments) representing eight different microscopic fields with over 200 cells. G Cell death in WT and cGAS −/− BMDMos that were first synchronized at G2/M, then γ‐irradiated (10 Gy) followed by release and analysis at indicated time points. Mean ± SD, x biological triplicates ( n = 3) per treatment group are shown. Data information: Statistical significance in (B), (C), and (F) was assessed using unpaired two‐tailed Student's t ‐test. *** P ≤ 0.001 and **** P ≤ 0.0001. Statistical significance in (G) was assessed using two‐way ANOVA test, **** P < 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Irradiation, Transfection, Plasmid Preparation, Staining, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Pulsed‐field gel electrophoresis analysis of γ‐irradiated (10 Gy) WT and cGAS −/− BMDMos. B, C Comet assay in GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells γ‐irradiated (IR: 10 Gy) for 15 min (B). RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293T cells stimulated with transfected DNA for 6 h (C). D, E Comet assay of HEK293 cells stimulated with 10 μg/ml cGAMP for indicate periods, then γ‐irradiated and incubated at 37°C for indicated duration (D). (E) Immunoblots of IRF3 phosphorylation in HEK293 cells treated as in (D). F–H Images (F) and quantifications (G) of comet tails 15 min after irradiation of GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells. RT–PCR analysis of IFNB1 response in GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells stimulated with transfected 23 DNA for 6 h (H). I, J Images (I) and quantifications (J) of micronuclei in GFP‐NLS‐ and GFP‐hcGASΔcGAMP‐expressing HEK293 cells 24 h after γ‐irradiation (IR; 10 Gy). DAPI (DNA). Scale bar: 10 μm. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. K Quantifications of comet tails 15 min after irradiation (10 Gy) of GFP‐NLS‐, GFP‐hcGAS‐, or GFP‐mcGAS‐expressing HEK293 cells. Each data set bar comet graph was calculated from six different microscopic fields with over 200 cells. Data information: Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. NS P > 0.05, *** P ≤ 0.001, and **** P ≤ 0.0001. Mean ± SEM of n = 3 independent experiments. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Pulsed-Field Gel, Electrophoresis, Irradiation, Single Cell Gel Electrophoresis, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Incubation, Western Blot, Phospho-proteomics

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A Reporter assays showing the effect of NLS and NES on cGAS‐mediated inhibition of DNA repair. B Both full‐length hcGAS and hcGAS cat (161–522aa) inhibit HR repair. C–E cGAS does not impede ATM activation. ATM phosphorylation in γ‐irradiated (10 Gy) GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293T cells (C), GFP‐NLS‐, GFP‐hcGAS‐, and GFP‐hcGASΔcGAMP‐expressing HEK293 cells (D), or γ‐irradiated (2.5 Gy) WT, cGAS −/− , and Sting −/− BMDMos (E). Data information: Data are means ± SD, n = 3. Statistical significance was assessed using one‐way ANOVA followed by Sidak's post‐test. *** P < 0.001 **** P < 0.0001, NS: P > 0.05. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Inhibition, Activation Assay, Phospho-proteomics, Irradiation, Expressing

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: A cGAS is not recruited to DSB sites: Confocal microscopic images of GFP‐NLS‐ or GFP‐hcGAS‐expressing U2OS‐DSB reporter cells incubated (or not) with Shield‐1 and 4‐OHT to induce the expression and translocation of mCherry‐LacI‐FokI (red) to specific DSB sites. Scale bar: 10 μm. The arrowheads indicate DSB sites. B cGAS does not co‐localize with γ‐H2AX at DSB sites: GFP‐NLS‐ or GFP‐hcGAS‐expressing HEK293 cells exposed (or not) to γ‐irradiation (IR: 10 Gy), then stained for γ‐H2AX. Scale bar: 10 μm. C, D Nuclear cGAS is mainly chromatin‐bound and remains unaltered upon γ‐irradiation. (C) Cytosolic (cyto) and nuclear fractions of γ‐irradiated (10 Gy, 30 min) BMDMos analyzed for cGAS and indicated molecules. (D) Cytosolic, soluble nuclear, and chromatin fractions from BMDMos were immunoblotted for cGAS and indicated proteins. E–G cGAS co‐isolates with DNA repair proteins because of bound chromatin bridges. (E) Nuclease digestion abrogates the co‐isolation of cGAS and DNA repair proteins: Lysates of control (−IR) and γ‐irradiated (+IR, 10 Gy, 30 min) GFP‐hcGAS‐expressing HEK293 cells were treated (or not) with benzonase before cGAS immunoprecipitation and analysis for indicated proteins. (F) Agarose gel analysis of DNA in corresponding cell lysates in (E). (G) Co‐isolation of cGAS and DNA repair proteins depends on its binding to DNA: cGAS pulldowns along with lysate inputs of control and γ‐irradiated HEK293 cells expressing GFP‐hcGAS or GFP‐hcGASΔDNA probed for indicated proteins. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Expressing, Incubation, Translocation Assay, Irradiation, Staining, Isolation, Control, Immunoprecipitation, Agarose Gel Electrophoresis, Binding Assay

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Confocal images of γ‐irradiated GFP‐NLS‐ and GFP‐hcGAS‐expressing HEK293 cells stained for RAD51 (red) with or without γ‐irradiation. Scale bar: 10 μm. Schematics of the D‐loop formation assay, including pre‐incubation of template dsDNA with cGAS cat (i) or with cGAS cat being added after RAD51 was bound to dsDNA (ii). Pre‐incubation of dsDNA with mcGAS cat prevents D‐loop formation by human RAD51, but does not affect the RAD1 activity once RAD51 filaments are bound to dsDNA. The percentage of D‐loop formed in each reaction (left) was graphed as the average of triplicates ± SD. Schematics of the D‐loop assay. Pre‐incubation of template dsDNA with hcGAS cat blocks subsequent D‐loop formation. The percentage of D‐loop formation (below) was graphed as the average of triplicates ± SD. Data information: Unpaired two‐tailed Student's t ‐test was used for statistical analyses. NS P > 0.05, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Irradiation, Expressing, Staining, Tube Formation Assay, Incubation, Activity Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: Chromatin‐bound cGAS is an inhibitor of DNA repair and hence accelerates genome destabilization and cell death

doi: 10.15252/embj.2019102718

Figure Lengend Snippet: Negative‐stain electron micrographs of cGAS‐dsDNA complexes following incubation of dsDNA with indicated cGAS variants. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation when pre‐incubated with dsDNA. Percentage of D‐loop formed in each reaction (left) graphed as the average of triplicates ± SD. Overview of a single 1:1 hcGAS‐DNA complex depicting the location of the Y215 within the cGAS‐dsDNA interface. DR‐GFP assay showing that hcGASΔDNA‐Y215E is impaired in HR inhibition. hcGASΔDNA‐Y215E but not hcGASΔcGAMP has a decreased affinity to dsDNA24. hcGASΔDNA‐Y215E and hcGASΔcGAMP are defective in synthase activity. Negative‐stain electron micrographs showing that hcGAScat‐ΔDNA‐Y215E is defective in inducing cGAS‐dsDNA complexes. Scale bar: 100 nm. Effect of indicated hcGAS variants on D‐loop formation. Data information: Data are means ± SD, n = 3. Unpaired Student's t ‐test was used for statistical analyses: NS P > 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Source data are available online for this figure.

Article Snippet: The anti‐p‐ATM (Ser1981), cGAS, and GFP antibody were from Santa Cruz.

Techniques: Staining, Incubation, Inhibition, Activity Assay

Journal:

Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

doi: 10.1091/mbc.E04-04-0294

Figure Lengend Snippet: Both Bax translocation and cytochrome c release induced by actinomycin D are inhibited in hFis1-depleted HeLa cells, whereas cytochrome c release, but not Bax translocation, is inhibited in Drp1-depleted cells. (A) HeLa cells depleted of hFis1 or Drp1 by RNAi along with control RNAi cells were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and double stained with anti-Bax (rabbit polyclonal, red) and anti-cytochrome c (mouse monoclonal, green) antibodies. (B) The number of cells displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of total cells counted in each RNAi cell population that had been treated with Act D and stained with anti-Bax and anti-cytochrome c (the same samples as shown in A). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (C) A unique group of cells (Bax had translocated but cytochrome c was not released) was seen in Drp1 RNAi cells. Four groups of cells were counted in each RNAi cell population that had been treated with Act D (the same samples as in A): i) Bax did not translocate and cytochrome c was not released; ii) Bax translocated, but cytochrome c is not released; iii) Bax not translocated, but cytochrome c is released; and iv) Bax translocated and cytochrome c is released were plotted as a percentage of total cells counted in each RNAi cell population. There were no cells categorized as iii, so only i, ii, and iv were plotted. Bars are labeled solid or striped, as in B. Data were plotted as the mean ± SD of at least three independent experiments. (D) HeLa cells depleted of Drp1 or hFis1 along with control RNAi were incubated with or without Act D (10 μM) for 8 h, fractionated into S and HM (mainly mitochondria), and analyzed for the distribution of Bax and cytochrome c by Western blotting. Fractionation quality was verified by the distribution of specific subcellular markers: CoxIV for mitochondria and actin for cytosol. To confirm the depletion of proteins (hFis1 and Drp1) and their localizations, hFis1 and Drp1 also were examined. The figure shown is representative of three independent experiments.

Article Snippet: The primary antibodies, their dilutions, and their sources are as follows: anti-Fis1 (1:500; Axxora, San Diego, CA), anti-Opa1 ( Zhu et al ., 2003 ), anti-DLP1/Drp1 (1:1000; BD Transduction Laboratories), anti-Bax (1: 1000; Santa Cruz Biotechnology), anti-cytochrome c (1:1000; BD Biosciences PharMingen), anti-PARP (1:1000; BIOMOL Research Laboratories, Plymouth Meeting, PA), and anti-actin (1:1000; Sigma-Aldrich, St. Louis, MO).

Techniques: Translocation Assay, Staining, Labeling, Incubation, Western Blot, Fractionation

Journal:

Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

doi: 10.1091/mbc.E04-04-0294

Figure Lengend Snippet: Depletion of Opa1 induces mitochondrial fragmentation, and cells become very sensitive to apoptosis. HeLa cells were transfected with pREP4 constructs containing shRNA of the target sequence of Opa1 or control, and the transfectants were selected by growing in media containing hygromycin B. (A) total cell lysates from the Opa1 RNAi cells along with control RNAi cells were prepared and the expression level of Opa1 was analyzed by Western blotting. Actin level also was analyzed as a loading control. (B) Mitochondria of control RNAi cells and Opa1 RNAi cells were visualized with Mitotracker Red CMXRos and analyzed by confocal microscopy. Enlargements are shown for detailed structure of mitochondria. (C) Percentage of cell population with fragmented (dotted), normal (solid), or elongated (striped) mitochondria in control RNAi or Opa1 RNAi culture. At least 200 cells in several fields were counted in each experiment. Data represent the mean ± SD of at least three independent experiments. (D) Opa1-depleted cells along with control RNAi cells were treated with STS (1 μM; 6 h), Act D (10 μM; 8 h), etoposide (100 μM; 30 h), or anti-Fas antibody (500 ng/ml; 15 h), and apoptotic nuclei (stained with Hoechst) were scored and plotted as a percentage of total cells (at least 200 cells) counted. Data are shown as the mean ± SD of three independent experiments. (E) Total DNA was isolated from the cells treated as described above and analyzed by agarose gel electrophoresis and visualized with ethidium bromide. The first lane is a DNA ladder control. The figure shown is a representative of three independent experiments. (F) HeLa cells depleted of Opa1 along with control RNAi cells were incubated with or without Act D (10 μM) for 8 h in the presence of zVAD-fmk, fixed, and stained with anti-Bax (red; our unpublished data) and anti-cytochrome c antibodies (green). (G) The number of cells in F displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of total cells counted in each RNAi cell population that had been treated with or without Act D and stained with anti-Bax and anti-cytochrome c antibodies (the same samples as shown in F). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (H) PARP cleavage was analyzed in the total extracts from HeLa cells depleted of Opa1 and control that were treated with STS (1 μM; 0, 3, 6 h), Act D (10 μM; 0, 4, 8 h), or anti-Fas (500 ng/ml; 0, 6, 15 h). The figure is a representative of at least three independent experiments.

Article Snippet: The primary antibodies, their dilutions, and their sources are as follows: anti-Fis1 (1:500; Axxora, San Diego, CA), anti-Opa1 ( Zhu et al ., 2003 ), anti-DLP1/Drp1 (1:1000; BD Transduction Laboratories), anti-Bax (1: 1000; Santa Cruz Biotechnology), anti-cytochrome c (1:1000; BD Biosciences PharMingen), anti-PARP (1:1000; BIOMOL Research Laboratories, Plymouth Meeting, PA), and anti-actin (1:1000; Sigma-Aldrich, St. Louis, MO).

Techniques: Transfection, Construct, shRNA, Sequencing, Expressing, Western Blot, Confocal Microscopy, Staining, Isolation, Agarose Gel Electrophoresis, Incubation, Translocation Assay

Journal:

Article Title: Roles of the Mammalian Mitochondrial Fission and Fusion Mediators Fis1, Drp1, and Opa1 in Apoptosis

doi: 10.1091/mbc.E04-04-0294

Figure Lengend Snippet: Cells depleted of both hFis1 and Opa1 show apoptosis resistance like hFis1-depleted cells. (A) HeLa cells depleted of hFis1, Opa1, or both of them by RNAi along with control RNAi cells were treated with STS (1 μM; 6 h), Act D (10 μM; 8 h), etoposide (100 μM; 30 h), or anti-Fas antibody (500 ng/ml; 15 h), and the nuclei were stained with Hoechst 33342 (1 μg/ml; 15 min at RT). Normal or apoptotic nuclei of these cells in several fields were counted under the fluorescent microscope (for UV excitation). At least 200 cells altogether in each treatment were counted and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data are shown as the mean ± SD of at least three independent experiments. (B) HeLa cells depleted of Opa1, hFis1, or both of them along with control RNAi cells were incubated with anti-Fas antibody (500 ng/ml) for the periods as indicated, and the nuclei were stained and counted as normal or apoptotic nuclei. At least 200 cells altogether were counted in each sample at each time point and plotted as a percentage of cells with apoptotic nuclei among the total cells counted. The data were plotted as the mean ± SD of at least three independent experiments. (C) Bax translocation and cytochrome c release induced by Act D treatment are inhibited in hFis/Opa1 RNAi cells. Left, HeLa cells depleted of both hFis1 and Opa1 by RNAi along with control RNAi cells were treated with Act D (10 μM; 8 h) in the presence of zVAD-fmk (50 μM), fixed, and double stained with anti-Bax (rabbit polyclonal, red) and anti-cytochrome c (mouse monoclonal, green) antibodies. Right, the number of cells displaying Bax translocation or cytochrome c release was counted and plotted as a percentage of the total cells counted in each RNAi cell population that had been treated with Act D and stained with anti-Bax and anti-cytochrome c antibodies (the same samples as shown on the left). At least 200 cells were counted altogether in several fields. Data are plotted as the mean ± SD of at least three independent experiments. (D) hFis1 depletion prevents mitochondrial membrane potential reduction induced by Opa1 depletion. Left, HeLa cells depleted of hFis1 (b), Opa1 (c), or both of them (d) along with control RNAi cells (a) were incubated with 5 μg/ml JC-1 for 20 min and observed by confocal microscopy. JC-1 is a cationic dye that indicates mitochondrial polarization by shifting its fluorescence emission from green to red. Regions of high mitochondrial membrane potential are indicated by red fluorescence, and regions of low mitochondrial membrane potential are indicated by green fluorescence. Right, the number of cells with mitochondria whose membrane potential was lost was counted and plotted as a percentage of the total cells counted in each RNAi cell population.

Article Snippet: The primary antibodies, their dilutions, and their sources are as follows: anti-Fis1 (1:500; Axxora, San Diego, CA), anti-Opa1 ( Zhu et al ., 2003 ), anti-DLP1/Drp1 (1:1000; BD Transduction Laboratories), anti-Bax (1: 1000; Santa Cruz Biotechnology), anti-cytochrome c (1:1000; BD Biosciences PharMingen), anti-PARP (1:1000; BIOMOL Research Laboratories, Plymouth Meeting, PA), and anti-actin (1:1000; Sigma-Aldrich, St. Louis, MO).

Techniques: Staining, Microscopy, Incubation, Translocation Assay, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Single S100A8 or S100A9 yeast transformants or (B) cotransformants with plasmids mCherry S100A8/ GFP S100A9 were grown overnight in SG, and images were obtained with a fluorescence microscope. (C) TCA precipitates of extracts from cells growing on glucose or galactose medium were separated by 10% SDS-PAGE and analyzed by Western blot.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Fluorescence, Microscopy, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: Fluorescent microscope images of GFP S100A8 and GFP S100A9 (green) after (A) 2 days or (B) 4 days of induction. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (C) Quantification of the percent of cells with GFP, GFP S100A8 or GFP S100A9 foci in the vacuole or in the cytoplasm, following 2 and 4 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: GFP (green) and mCherry (red) fluorescent microscope images of mCherry S100A8/ GFP S100A9 cotransformed cells after 2 or 4 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Microscopy

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Confocal images of GFP, GFP S100A8 or GFP S100A9 (green) after 2 days of induction in pep4Δ mutants cells. Lipophilic dye FM4-64 was used to visualize vacuoles (red). (B) Quantification of the percent of cells with GFP, GFP S100A8 or GFP S100A9 foci in the vacuole of pep4 Δ cells, following 2 days of induction.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques:

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) NI, noninduced control (Lanes 1, 4, 7, 10). Extracts of cells induced for 2 or 4 days to produce GFP S100A8 (Lanes 2 and 3), GFP S100A9 (Lanes 5 and 6) and mCherry S100A8/ GFP S100A9 (Lanes 8, 9 and 11,12) were separated on a native gel and analyzed by Western blot. (B) Semi-denaturing agarose detergent gel. After 2 days of induction GFP S100A8 (Lane 1), GFP S100A9 (Lane 3) or cotransformants mCherry S100A8/ GFP S100A9 (Lanes 7, 8 and 10, 11) formed SDS-stable aggregates in yeast cells. Boiling (+) the samples led to full soloubilization of aggregates to the monomeric form (Lanes 2 and 4). Total cell extracts (180 µg) were resolved using SDD-AGE. Blots were probed with anti-GFP or mCherry antibodies. Total cell extract of Q103 GFP cells (90 µg) was prepared after 24 h of induction (lane 5). (C) Filter retardation assay of cells grown for 3 and 5 days under inducing conditions. Loading control was visualized by CBB staining. Empty vector-transfected cells were used as control. (D) Spheroplasts of control and induced cells stained with ThT after 3 days of incubation on galactose plates.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Control, Western Blot, Staining, Plasmid Preparation, Transfection, Incubation

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Ten-fold dilutions of yeast cells transformed with p GFP-S100A8 , p GFP-S100A9 or both plasmids were plated on glucose (non-inducing) or galactose (inducing) plates and photographed after 72 h. (B) Non-tagged proteins as in A.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Transformation Assay

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) wild type and cdc53-1 ts mutant cells expressing p YES2-S100A8 or p YES2-S100A9 were spotted on glucose or galactose plates and photographed after 72 h. (B ) Semi-denaturing agarose gel. After 2 days of induction GFP S100A9 forms aggregates in wild type and ts strain cdc53-1 at 30°C and 32°C. Total cell extracts (180 µg) were resolved using SDD-AGE. (C ) Ten-fold dilutions of cdc53-1 , cdc34-2 , srp1-31, and sec27-1 yeast cells transformed with p TET-S100A8 or p TET-S100A9 were spotted on SD plates with (inducing) or without (non-inducing) 5 µg/ml doxycycline and photographed after 72 h.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Mutagenesis, Expressing, Agarose Gel Electrophoresis, Transformation Assay

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: (A) Viability of wild type or hsp104Δ yeast in the presence of S100A8, S100A9 and S100A8/9 proteins. p YES2-S100A8 , p YES2-S100A9 or both plasmids were expressed in wild type or hsp104Δ mutant cells. Viability was monitored using the spot test assay on inducing (galactose) or noninducing (glucose) plates. (B) Ten-fold dilutions of wild type cells or hsp104Δ mutants transformed with p GALSc104(WT) and with p YES2-S100A8, p YES2-S100A9 or both S100 plasmids were plated on glucose (non-inducing) or galactose (inducing) plates. (C) Confocal images of GFP S100A8 and GFP S100A9 after 2 days of induction in wild type or Δhsp104 mutant cells. (D) Cell extracts were prepared from wild type or hsp104Δ mutant cells expressing p GFP-S100A8 or p GFP-S100A9 after 2 days of induction. Extracts were incubated in 2% SDS sample buffer with (+) or without (−) boiling, loaded on agarose gels, and analyzed by Western blot using anti-GFP antibodies to detect the S100A8 and S100A9 proteins.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Mutagenesis, Spot Test, Transformation Assay, Expressing, Incubation, Western Blot

Journal: PLoS ONE

Article Title: Aggregation of Human S100A8 and S100A9 Amyloidogenic Proteins Perturbs Proteostasis in a Yeast Model

doi: 10.1371/journal.pone.0058218

Figure Lengend Snippet: Cells expressing empty vector (p YES2), p YES2-S100A8 , p YES2-S100A9 , or cotransformants with p YES2-S100A8/ p YES2-S100A9 in sse1, sse2, ssa1, ssa2, ssa3, hsp26, and ydj1 mutants and the isogenic wild type parent were spotted on galactose and glucose plates and photographed after 72 h.

Article Snippet: For detecting non-tagged proteins, we used monoclonal and polyclonal anti-calgranulin A (S100A8) and anti-calgranulin B (S100A9) (Santa Cruz Biotechnology) at 1∶100 dilution.

Techniques: Expressing, Plasmid Preparation

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Evolutionary conservation and sequence homology analysis of calreticulin across multiple species. ( A ) Phylogenetic analysis of calreticulin amino acid sequences from different species. The neighbor-joining phylogenetic tree was constructed using the bootstrap method in MEGA version 10.2.2 with 1000 bootstrap replicates. ( B ) Sequence homology analysis of calreticulin amino acid sequences among various species. The matrix shown represents a bidirectional pairwise comparison of calreticulin sequences. The upper triangular region displays the percentage of sequence identity (% identity), while the lower triangular region shows the corresponding percentage of sequence divergence (% divergence) between each pair of species. Black squares along the diagonal indicate self-alignments, where sequence identity is 100% and divergence is 0%.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Sequencing, Construct, Comparison

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Distribution of calreticulin in different goat organs. ( A ) Immunohistochemical staining illustrating the localization of calreticulin in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. Bars = 50 μm. ( B ) Analysis of calreticulin protein expression levels via Western blot in the nasal mucosa, pharynx, trachea, lung, heart, liver, spleen, and small intestine of goats. ( C ) The band obtained by Western blot was analyzed by gray value, and the result was expressed as the gray value ratios of the CALR/GAPDH. All data shown are the mean ± SD from three independent experiments. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Immunohistochemical staining, Staining, Expressing, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Expression and purification of recombinant goat calreticulin using the Pichia pastoris expression system. ( A ) Schematic representation of the recombinant plasmid PpIC9K-CALR. ( B ) Agarose gel electrophoresis was performed to analyze the original recombinant plasmid (lane 1), the plasmid digested with EcoRI alone (lane 2), and the plasmid digested with both EcoRI and NotI (lane 3). ( C ) SDS-PAGE analysis of recombinant calreticulin expression in methanol-induced positive transformants, stained with Coomassie Brilliant Blue: Lane 1—culture supernatant of methanol-induced transformants; Lane 2—flow-through during Ni-NTA affinity purification; Lane 3—wash fraction; Lane 4—eluted protein with 200 mM imidazole; Lane 5—desalted and concentrated protein following ultrafiltration. ( D ) Western blot analysis confirming the identity of purified recombinant goat calreticulin (lane order as in panel ( C )). ( E ) SEC-HPLC analysis of the molecular weight and purity of yeast-secreted recombinant goat calreticulin. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Expressing, Purification, Recombinant, Plasmid Preparation, Agarose Gel Electrophoresis, SDS Page, Staining, Affinity Purification, Western Blot, Molecular Weight

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Antibacterial activity of recombinant goat calreticulin. ( A – C ) CFU assays were performed to evaluate the inhibitory effects of calreticulin against Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( D – F ) Growth curve analysis of Escherichia coli , Salmonella typhimurium , and Pasteurella multocida in the presence of calreticulin to assess its impact on bacterial proliferation. All data are presented as mean ± SD from three independent experiments. Statistical significance was determined using one-way ANOVA. ns, no significance; *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Activity Assay, Recombinant

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Recombinant goat calreticulin binds to and agglutinates bacteria. ( A – C ) ELISA analysis of bacterial binding: Escherichia coli , Salmonella typhimurium , and Pasteurella multocida (1 × 10 7 CFU/mL) were immobilized on microtiter plates and incubated with either CaCl 2 (10 mM), His-tag peptide (100 μg/mL), His-tag peptide (100 μg/mL) + CaCl 2 (10 mM), calreticulin (100 μg/mL), or calreticulin (100 μg/mL) + CaCl 2 (10 mM). Binding of calreticulin was detected using anti-His tag antibodies. ( D ) Western blot analysis of bacterial pellets after incubation with calreticulin (100 μg/mL) to confirm binding to Escherichia coli , Salmonella typhimurium , and Pasteurella multocida . ( E ) ELISA analysis of calreticulin binding activity to LPS. ( F ) Log-phase Escherichia coli , Salmonella typhimurium , and Pasteurella multocida were labeled with CFSE and incubated with calreticulin (100 μg/mL) ± 10 mM Ca 2+ at 37 °C for 2 h. Bacterial agglutination was visualized by fluorescence microscopy. Bars = 100 μm. All data are expressed as the mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. ns, no significance; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Recombinant, Bacteria, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Western Blot, Activity Assay, Labeling, Agglutination, Fluorescence, Microscopy

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal infection with Pasteurella multocida upregulates calreticulin expression in the respiratory tissues of the lambs. Thirty-day-old lambs were intranasally challenged with Pasteurella multocida and euthanized 24 h post-infection, after which respiratory tract tissues, including the nasal cavity, trachea, and lung, were collected for further analysis. ( A ) Immunohistochemical staining showing calreticulin expression in the nasal mucosa, trachea, and lungs following infection. Bars = 20 μm. ( B ) Quantitative analysis of immunohistochemical staining based on gray value measurements. ( C ) RT-qPCR analysis of calreticulin mRNA expression in different respiratory tissues after Pasteurella multocida infection. ( D ) Western blot analysis of calreticulin protein levels in infected respiratory tissues. ( E ) Densitometric analysis of Western blot results. All data are presented as mean ± SD from three independent experiments. Statistical significance was evaluated using one-way ANOVA. ** p < 0.01; *** p < 0.001. Original Western blot images can be found in .

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Expressing, Immunohistochemical staining, Staining, Quantitative RT-PCR, Western Blot

Journal: Biomolecules

Article Title: Identification of a Novel Antibacterial Function of Mammalian Calreticulin

doi: 10.3390/biom15070966

Figure Lengend Snippet: Intranasal administration of calreticulin alleviates Pasteurella multocida -induced pathological injury and promotes bacterial clearance in the lambs. Thirty-day-old lambs were intranasally infected with Pasteurella multocida ; 6 h post-infection, 1 mL of recombinant calreticulin (2.5 mg/mL) was administered intranasally. Lambs were euthanized and necropsied at 24 h post-infection for sample collection. ( A ) Histopathological evaluation of nasal mucosa ( a – c ), trachea ( d – f ), and lung tissues ( g – i ) using H&E staining. Bars = 20 μm. ( B ) Average injury scores calculated for all lambs ( n = 3 replicates/group). ( C ) In situ hybridization with a Pasteurella multocida -specific fluorescent probe to detect bacterial load in nasal cavity ( a , b ), tracheal ( c , d ), and lung ( e , f ) tissue sections. Bars = 100 μm. ( D ) Quantification of fluorescence intensity derived from in situ hybridization results. All data are presented as mean ± SD from three independent experiments. Statistical significance was assessed using one-way ANOVA. *** p < 0.001.

Article Snippet: Antibodies: GAPDH antibody (Proteintech, Wuhan, China, 60004-1-Ig, Western blot, 1:1000); calreticulin antibody (Proteintech, 10292-1-AP, Western blot, 1:1000; IHC, 1:200); His-tag (Proteintech, 66005-1-Ig, Western blot, 1:10,000).

Techniques: Infection, Recombinant, Staining, In Situ Hybridization, Fluorescence, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Nuclear Localization and Chromatin Targets of p21-activated Kinase 1

doi: 10.1074/jbc.m412607200

Figure Lengend Snippet: FIG. 1. Nuclear Localization of Pak1 and identification of nuclear lo- calizing sequence motifs. A, proteins from different subcellular locations of MCF-7 cells were isolated by sequential extraction and were analyzed by SDS- polyacrylamide gel electrophoresis fol- lowed by Western blot analysis with Pak1-specific antibody. Poly(ADP-ribose) polymerase (PARP), Paxillin, and Lamin B1 were used as control markers for chro- matin, cytoplasm, and nuclear matrix, re- spectively. B, confocal microscopy pic- tures of MCF-7 cells show the subcellular localization of transiently transfected Myc-tagged Pak1 WT protein (EGF) and different Pak1 NLS mutants (Mut) treated with EGF. Cells were treated with 100 ng of human EGF/ml medium for 45 min, and the staining was done as de- scribed under “Materials and Methods.” The best representative picture of each group is shown. The three nuclear local- izing signals (NLS1, NLS2, and NLS3) and their positions on Pak1 protein are shown schematically. C, 50 transiently transfected cells were checked for local- ization of Pak1 (WT and NLS mutants). The percentages of cells with nuclear lo- calization were calculated and are shown as a histogram.

Article Snippet: The purity of the isolated fractions was established by Western blot analysis with antibodies against marker proteins like, poly(ADP-ribose) polymerase (Pharmingen) for chromatin, Lamin B1 (Biotechnology Inc., Santa Cruz, CA) for nuclear matrix and Paxillin (BD Transduction Laboratories) for cytoplasm.

Techniques: Sequencing, Isolation, Extraction, Polyacrylamide Gel Electrophoresis, Western Blot, Control, Confocal Microscopy, Transfection, Staining

Journal: Journal of Virology

Article Title: The Mdm2 Ubiquitin Ligase Enhances Transcriptional Activity of Human Papillomavirus E2

doi: 10.1128/jvi.01551-08

Figure Lengend Snippet: FIG. 3. Mdm2 interacts with 16E2 in vitro and in vivo. (a) In vitro-translated and radiolabeled Mdm2 was incubated with bacterially purified GST-tagged E2. GST alone and GST-p53 were included as negative and positive controls, respectively. Bound proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The Coomassie blue stains of the GST inputs are also included in the top panel. An asterisk indicates the full-length GST fusion proteins. (b) Mdm2 binds to the C-terminal region of E2. GST-16E2 and a number of GST-tagged fragments of E2 (right) were incubated with in vitro-translated and radiolabeled Mdm2, and bound proteins were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Fifty percent of the input is included, and the GST inputs are shown in the bottom panel and are stained with Coomassie blue. An asterisk indicates the full-length GSTs. NE2, N-terminal half of E2 protein; CE2, C-terminal half of E2 protein. (c) E2 and Mdm2 bind in vivo. 293 cells were transfected using either Mdm2 alone or Mdm2 with GFP-tagged E2. Cell extracts were immunoprecipitated (IP) using polyclonal anti-GFP antibodies, followed by Western blot analysis using antibodies against Mdm2 or GFP. An asterisk indicates nonspecific bands. -ve, untransfected cells.

Article Snippet: At 24 h posttransfection, E2 was immunoprecipitated from cell extracts, using antibodies against green fluorescent protein (GFP) (polyclonal; Santa Cruz), for 3 to 4 h. After extensive washing, the immunoprecipitate was analyzed by Western blotting, using an anti-Mdm2 monoclonal antibody (kindly provided by Giannino Del Sal).

Techniques: In Vitro, In Vivo, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography, Staining, Transfection, Immunoprecipitation, Western Blot

Journal: Science Advances

Article Title: Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis

doi: 10.1126/sciadv.aat3834

Figure Lengend Snippet: ( A and B ) TF1.8 cells were starved overnight in the presence of 0.5% FCS, preincubated with either vehicle or 280 nM ruxolitinib for 10 min, and stimulated with IL-3 (50 ng/ml) for different times. Cells were lysed and subjected to immunoprecipitation with (A) anti-phosphotyrosine (4G10) or (B) anti-phosphorylated JAK2 (Y1007/1008) antibodies, followed by immunoblotting with JAK2 and JAK1 antibodies. As a control, total lysates from the same experiment were immunoblotted with p-JAK1, p-JAK2, JAK1, and JAK2 antibodies as indicated. ( C ) TF1.8 cells were starved overnight in the presence of 0.5% FCS, preincubated with either vehicle or 280 nM ruxolitinib for 10 min, and stimulated with different doses of IL-3 for 5 min. Cells were lysed and subjected to immunoprecipitation (IP) with anti-phosphotyrosine (4G10) antibody, followed by immunoblotting with JAK1 and JAK2 antibodies. As a control, total lysates from the same experiment were immunoblotted with p-JAK1, p-JAK2, JAK1, and JAK2 antibodies. ( D ) SET-2 cells were incubated in 0.5% FCS for 6 hours before the addition of 280 nM ruxolitinib for 0, 10, or 30 min or stimulation with IL-3 (50 ng/ml) and EPO for 5 min. Cells were lysed and subjected to immunoprecipitation with anti-phosphotyrosine (4G10) antibody, followed by immunoblotting with JAK2 and STAT5 antibodies. As a control, total lysates from the same experiment were immunoblotted with p-JAK2, p-STAT5, JAK2, and actin antibodies. ( E ) Recombinant JAK2 kinase domain was mixed with recombinant tyrosine phosphatase PTP1B and ruxolitinib or no inhibitor in phosphatase assay buffer. Phosphatase reactions were incubated at room temperature for 0, 2, 5, and 20 hours, fractionated by SDS–polyacrylamide gel electrophoresis (PAGE), and immunoblotted with p-JAK2 and JAK2 antibody. ( F ) TF1.8 cells were starved overnight in the presence of 0.5% FCS, preincubated for 10 min with MG132 plus either vehicle or 280 nM ruxolitinib, and stimulated with IL-3 (50 ng/ml) for 0, 5, or 10 min. Cells were lysed and subjected to immunoprecipitation with JAK2 antibody, followed by immunoblotting with ubiquitin antibody conjugated to horseradish peroxidase (αUb-HRP) or p-JAK2 antibody. ( G ) Mononuclear cells from patient with myelofibrosis RAH1 were cultured in 10% FCS with EPO and IL-3 (1 ng/ml each) and 280 nM ruxolitinib or DMSO for 12 hours. Cells were then washed in cold RPMI and cultured in MG132 without additives for 5 or 15 min. Cells were lysed and subjected to immunoprecipitation with JAK2 antibody, followed by immunoblotting with ubiquitin-HRP or JAK2 antibody.

Article Snippet: A primary antibody against TYK2 (#SC-169) and ubiquitin-HRP conjugate (SC-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Western Blot, Control, Incubation, Recombinant, Phosphatase Assay, Polyacrylamide Gel Electrophoresis, Ubiquitin Proteomics, Cell Culture

Journal: Science Advances

Article Title: Accumulation of JAK activation loop phosphorylation is linked to type I JAK inhibitor withdrawal syndrome in myelofibrosis

doi: 10.1126/sciadv.aat3834

Figure Lengend Snippet: ( A ) SET-2 cells were treated with ruxolitinib or DMSO for 12 hours, followed by washout as described in . Whole-cell lysates were prepared and subjected to immunoprecipitation with human βc receptor antibody and immunoblotted with phosphotyrosine, p577Y βc, and βc antibodies. ( B ) γ2A cells that are JAK2 deficient were stably transfected with βc and IL3Rα receptors (γ2A/IL3Rα/βc) and then additionally stably transfected with either JAK2 WT -FLAG or JAK2 KI -FLAG. After overnight starvation, cells were pretreated with 150 nM of the type I inhibitor fedratinib and stimulated for 0, 5, 30, or 60 min with IL-3 (25 ng/ml). Cells were lysed and subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with p-JAK2 and JAK2 antibodies. ( C ) Recombinant JAK1 kinase domain was mixed with recombinant tyrosine phosphatase PTP1B and ruxolitinib or no inhibitor in phosphatase assay buffer. Phosphatase reactions were incubated at room temperature for 0, 2, 5, and 20 hours, fractionated by SDS-PAGE, and immunoblotted with p-JAK1 antibody. Coomassie blue staining was used as a loading control. ( D ) TF1.8 cells were starved overnight in 0.5% FCS and then treated with DMSO, 280 nM ruxolitinib, or 150 nM fedratinib for 10 min before stimulation with IL-3 (50 ng/ml) for 0 or 5 min. Whole-cell lysates were prepared and subjected to immunoprecipitation with anti-JAK1 antibody, followed by immunoblotting with ubiquitin-HRP or JAK1 antibody. ( E ) γ2A/IL3Rα/βc cells were stably transfected with either JAK2 WT -FLAG or JAK2 KI -FLAG. After overnight starvation, cells were pretreated with DMSO or 11 nM itacitinib and stimulated for 5 min with IL-3 (50 ng/ml). Cells were lysed and subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with p-JAK2 and JAK2 antibodies. ( F ) γ2A/IL3Rα/βc cells expressing JAK2 KI -FLAG were transfected with control small interfering RNA (siRNA) or JAK1 siRNA. After overnight starvation, cells were pretreated with fedratinib for 10 min and stimulated with IL-3 (50 ng/ml). Cells were lysed and subjected to immunoprecipitation with anti-FLAG antibody, followed by immunoblotting with p-JAK2 and JAK2 antibodies. As a control, total lysates from the same experiment were immunoblotted with JAK1 and actin antibodies.

Article Snippet: A primary antibody against TYK2 (#SC-169) and ubiquitin-HRP conjugate (SC-8017) were purchased from Santa Cruz Biotechnology.

Techniques: Immunoprecipitation, Stable Transfection, Transfection, Western Blot, Recombinant, Phosphatase Assay, Incubation, SDS Page, Staining, Control, Ubiquitin Proteomics, Expressing, Small Interfering RNA