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  • 99
    New England Biolabs exonuclease i
    Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. ( a , b ) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a 32 P-labeled telomere probe (TTAGGG) 3 . Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a . Representative gels of the TRF assay are shown in b . ( c ) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. ( d ) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of <t>Exonuclease</t> I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the 32 P-labeled (CCCTAA) 3 probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. ( e ) Overhang signals for each cell line from d were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3). P- values were obtained using the Student’s t -test. ** P
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    99
    Thermo Fisher nacl
    Liquid droplet characteristics of <t>p‐tau441</t> Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high <t>NaCl</t> concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.
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    94
    GE Healthcare shrimp alkaline phosphatase
    Liquid droplet characteristics of <t>p‐tau441</t> Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high <t>NaCl</t> concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.
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    99
    Thermo Fisher bsa
    ). (D) ELISA sugar-binding assay for <t>PdCoV</t> S1-NTD. Here, the ELISA plates were precoated with sugar-rich mucin, and then PdCoV S1-NTD was added and incubated with mucin. Mucin-bound S1-NTD was detected using antibodies recognizing its C-terminal His 6 tag. Porcine epidemic diarrhea virus (PEDV) S1 was used as the positive control. PdCoV S1-CTD, SARS-CoV S1-CTD, and <t>BSA</t> were used as negative controls. A plate without mucin was used as an additional negative control. Statistical analyses were performed using two-tailed t test. Error bars indicate standard errors of the means (SEM) ( n = 5). ***, P
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    95
    GE Healthcare exonuclease i
    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
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    99
    Thermo Fisher taq dna polymerase
    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
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    Thermo Fisher platinum taq polymerase
    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
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    Thermo Fisher decalabel dna labeling kit
    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
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    96
    GE Healthcare alkaline phosphatase
    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
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    Thermo Fisher bigdye terminator v3 1 cycle sequencing kit
    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and <t>exonuclease</t> I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.
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    97
    TaKaRa random primer dna labeling kit
    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Thermo Fisher dna polymerase
    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Thermo Fisher buffer ii
    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Thermo Fisher shrimp alkaline phosphatase
    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) <t>DNA</t> sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) <t>GFP</t> expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.
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    Image Search Results


    Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. ( a , b ) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a 32 P-labeled telomere probe (TTAGGG) 3 . Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a . Representative gels of the TRF assay are shown in b . ( c ) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. ( d ) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of Exonuclease I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the 32 P-labeled (CCCTAA) 3 probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. ( e ) Overhang signals for each cell line from d were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3). P- values were obtained using the Student’s t -test. ** P

    Journal: Cell Discovery

    Article Title: Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells

    doi: 10.1038/celldisc.2017.34

    Figure Lengend Snippet: Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. ( a , b ) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a 32 P-labeled telomere probe (TTAGGG) 3 . Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a . Representative gels of the TRF assay are shown in b . ( c ) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. ( d ) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of Exonuclease I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the 32 P-labeled (CCCTAA) 3 probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. ( e ) Overhang signals for each cell line from d were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3). P- values were obtained using the Student’s t -test. ** P

    Article Snippet: Genomic DNA was digested withHin fI and Rsa I (New England Biolabs, Ipswich, MA, USA) for 16 h. In all, 5 μg each of the digested DNA was then incubated with and without 20 U of Exonuclease I (New England Biolabs) for 6 h. The reaction mixtures were fractionated on a 1.2% agarose gel in 1× Tris/borate/EDATA (TBE) buffer for 1.5 h at 60 V and dried on a gel dryer at 50 °C for 2 h. The dried gel was pre-hybridized in hybridization buffer (0.5 m Na2 HPO4 pH 7.2, 1 mM EDTA, 7% sodium dodecyl sulfate) and hybridized in fresh buffer with a32 P-labeled C-strand probe (CCCTAA)3 .

    Techniques: Labeling, TRF Assay, Staining, DNA Extraction, Hybridization

    Liquid droplet characteristics of p‐tau441 Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.

    Journal: The EMBO Journal

    Article Title: Tau protein liquid–liquid phase separation can initiate tau aggregation

    doi: 10.15252/embj.201798049

    Figure Lengend Snippet: Liquid droplet characteristics of p‐tau441 Qualitative distribution of phosphorylation sites in p‐tau441 [pS68/69, pT153, pT175, pT181, pS184, pS199, pS202, pT205, pS210, pT212, pS214, pT217, pT231, pS235, pS262, pS324, pY310, pS316, pS396, pS404, pS422 (Mair et al )]. The charge at pH 7.4 of domains in unphosphorylated tau441 is indicated as well. Liquid–liquid phase separation (LLPS) of p‐tau441 in presence of molecular crowding (12.5% w/v Ficoll‐400). No phase separation is observed without crowding agent or in the absence of p‐tau441 protein. Liquid droplets formed by p‐tau441 in the presence of 10% (w/v) PEG were negative stained with uranyl‐acetate and visualized by transmission electron microscopy (TEM). p‐tau441 droplets are decorated with gold particles after immunogold labeling using anti‐tau antibody K9JA. . p‐tau441 droplets (in buffer with 10% PEG) exhibit glass surface wetting properties characteristics for liquids. Phase diagram of tau LLPS (p‐tau441 concentration (μM) versus PEG concentration (% w/v). In conditions modeling the intraneuronal environment (∼2 μM tau, 10% PEG, pH 7.5), p‐tau441 droplets can form at very high NaCl concentrations (up to 3 M NaCl) in the buffer. Guanidinium hydrochloride (GdmHCl) prevents p‐tau441 LLPS at 3 M; at this concentration, tau aggregates become visible. The chaotropic salt sodium thiocyanate (NaSCN) can inhibit LLPS with increasing concentration, whereas droplets become larger in the presence of the cosmotropic salt ammonium sulfate ((NH 4 ) 2 SO 4 ). Urea, which denatures proteins by unfolding secondary structures, prevents p‐tau441 LLPS. n = 3 per condition, determined after 3 h. Brightfield images for p‐tau441 LLPS under different salt conditions graphed in (G). The addition of 10% 1,6‐hexanediol to p‐tau441 droplets substantially reduced the amount of tau droplets formed. Data information: In (F) and (G), the phase diagrams show the average of three measurements.

    Article Snippet: Excess dye was removed by dialysis (SlideLizer MWCO 10 kDa, Pierce) against (10 mM HEPES buffer, 20 mM NaCl, pH 7.4) at 4°C overnight. p‐tau441‐a568 was produced by labeling p‐tau441 using thiol‐reactive AlexaFluor568 C5 maleimide (Life Technologies) according to the manual.

    Techniques: Staining, Transmission Assay, Electron Microscopy, Transmission Electron Microscopy, Labeling, Concentration Assay

    In vitro phase separation of tau initiated by crowding agents LLPS of p‐tau441 and p‐tau256 can also be initialized using crowding agent PEG‐8000 or a combination of PEG‐8000 with bovine serum albumin (BSA), whereas the soluble control protein GFP did not undergo LLPS in the presence of 10% PEG. We estimated the concentration of fluorescently labeled p‐tau441‐Alexa568 (10% PEG, 50 mM NaCl, 5 μM p‐tau441‐a568) in the droplets by confocal imaging ( z = 2 μm) of 30‐min‐old droplets. The measured maximum droplet fluorescence intensity was calibrated against different concentrations of p‐tau441‐Alexa568 in solution without LLPS initiation (no PEG, 50 mM NaCl). Cross‐sectional profile along the white arrow visualizes the intensity levels of droplets. The mean of the detected p‐tau441‐a568 concentration was 34.3 ± 6.4 μM in the droplets and 3.6 ± 0.3 μM in the solution phase. This value might be underestimating the actual tau concentration in the droplets because fluorophore:fluorophore quenching and other artifact resulting from the highly viscous and crowded environment in the droplets are not considered. Absorbance spectra of free and p‐tau441 bound Alexa568 shows differences in the intensity but no shift in the wavelengths. The intensity of free Alexa568 dye imaged at 533 nm in confocal microscopy did not change with the amount of PEG in solution. By confocal imaging, the crowding agent dextran remains excluded from p‐tau441 droplets initiated by adding 9% dextran‐70 kDa and 1% of fluorescent dextrans of different molecular weights. The addition of methylene blue, viscous aqua, and ThioS, all dyes with affinity to hydrophobic protein areas, to freshly prepared p‐tau441 droplets reveals the co‐partitioning and retention of these dyes in the droplets. The fluorescence may be further enhanced by inhibition of free rotation of the dyes due to the higher droplet viscosity. Data information: In right graphs of (B, C), data are represented as mean ± s.d.

    Journal: The EMBO Journal

    Article Title: Tau protein liquid–liquid phase separation can initiate tau aggregation

    doi: 10.15252/embj.201798049

    Figure Lengend Snippet: In vitro phase separation of tau initiated by crowding agents LLPS of p‐tau441 and p‐tau256 can also be initialized using crowding agent PEG‐8000 or a combination of PEG‐8000 with bovine serum albumin (BSA), whereas the soluble control protein GFP did not undergo LLPS in the presence of 10% PEG. We estimated the concentration of fluorescently labeled p‐tau441‐Alexa568 (10% PEG, 50 mM NaCl, 5 μM p‐tau441‐a568) in the droplets by confocal imaging ( z = 2 μm) of 30‐min‐old droplets. The measured maximum droplet fluorescence intensity was calibrated against different concentrations of p‐tau441‐Alexa568 in solution without LLPS initiation (no PEG, 50 mM NaCl). Cross‐sectional profile along the white arrow visualizes the intensity levels of droplets. The mean of the detected p‐tau441‐a568 concentration was 34.3 ± 6.4 μM in the droplets and 3.6 ± 0.3 μM in the solution phase. This value might be underestimating the actual tau concentration in the droplets because fluorophore:fluorophore quenching and other artifact resulting from the highly viscous and crowded environment in the droplets are not considered. Absorbance spectra of free and p‐tau441 bound Alexa568 shows differences in the intensity but no shift in the wavelengths. The intensity of free Alexa568 dye imaged at 533 nm in confocal microscopy did not change with the amount of PEG in solution. By confocal imaging, the crowding agent dextran remains excluded from p‐tau441 droplets initiated by adding 9% dextran‐70 kDa and 1% of fluorescent dextrans of different molecular weights. The addition of methylene blue, viscous aqua, and ThioS, all dyes with affinity to hydrophobic protein areas, to freshly prepared p‐tau441 droplets reveals the co‐partitioning and retention of these dyes in the droplets. The fluorescence may be further enhanced by inhibition of free rotation of the dyes due to the higher droplet viscosity. Data information: In right graphs of (B, C), data are represented as mean ± s.d.

    Article Snippet: Excess dye was removed by dialysis (SlideLizer MWCO 10 kDa, Pierce) against (10 mM HEPES buffer, 20 mM NaCl, pH 7.4) at 4°C overnight. p‐tau441‐a568 was produced by labeling p‐tau441 using thiol‐reactive AlexaFluor568 C5 maleimide (Life Technologies) according to the manual.

    Techniques: In Vitro, Concentration Assay, Labeling, Imaging, Fluorescence, Confocal Microscopy, Inhibition

    ). (D) ELISA sugar-binding assay for PdCoV S1-NTD. Here, the ELISA plates were precoated with sugar-rich mucin, and then PdCoV S1-NTD was added and incubated with mucin. Mucin-bound S1-NTD was detected using antibodies recognizing its C-terminal His 6 tag. Porcine epidemic diarrhea virus (PEDV) S1 was used as the positive control. PdCoV S1-CTD, SARS-CoV S1-CTD, and BSA were used as negative controls. A plate without mucin was used as an additional negative control. Statistical analyses were performed using two-tailed t test. Error bars indicate standard errors of the means (SEM) ( n = 5). ***, P

    Journal: Journal of Virology

    Article Title: Cryo-Electron Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State

    doi: 10.1128/JVI.01556-17

    Figure Lengend Snippet: ). (D) ELISA sugar-binding assay for PdCoV S1-NTD. Here, the ELISA plates were precoated with sugar-rich mucin, and then PdCoV S1-NTD was added and incubated with mucin. Mucin-bound S1-NTD was detected using antibodies recognizing its C-terminal His 6 tag. Porcine epidemic diarrhea virus (PEDV) S1 was used as the positive control. PdCoV S1-CTD, SARS-CoV S1-CTD, and BSA were used as negative controls. A plate without mucin was used as an additional negative control. Statistical analyses were performed using two-tailed t test. Error bars indicate standard errors of the means (SEM) ( n = 5). ***, P

    Article Snippet: The membranes were dried and blocked with 1% BSA and then incubated with 1 μM PdCoV S1-CTD at 4°C for 2 h. After washes with PBS buffer, the membranes were incubated with anti-His6 antibody (Life Technologies Inc.) at 4°C for 2 h, washed with PBS, incubated with HRP-conjugated goat anti-mouse IgG antibody (1:5,000) at 4°C for 2 h, and washed with PBS.

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Positive Control, Negative Control, Two Tailed Test

    Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and exonuclease I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.

    Journal: Genome Research

    Article Title: A Microsphere-Based Assay for Multiplexed Single Nucleotide Polymorphism Analysis Using Single Base Chain Extension

    doi:

    Figure Lengend Snippet: Schematic presentation of the microsphere-based single base chain extension assays. DNA fragments containing the polymorphic site to be typed were amplified either individually or by multiplexed PCR (step 1 ). The PCR products containing a SNP site were pooled and treated with SAP and exonuclease I (step 2 ). After heat inactivation of the enzymes, the PCR products were used in the SBCE reactions (step 3 ) as described in Methods. For every SNP, one capture oligonucleotide probe with a unique ZipCode sequence was designed and used to assay the two alleles in each of two separate wells with a different labeled ddNTP per well. Multiplexed SNP analysis could be achieved by the employment of different ZipCode sequences for different SNPs in the presence of pooled PCR products. After the completion of the SBCE reaction, ∼1200 of each type of microsphere [with an attached oligonucleotide encoding the complement to the ZipCode sequences and a common luciferase sequence (SeqLUC)] were added to the completed SBCE reactions. The hybridization reactions were carried out at 40°C in the presence of NaCl for > 2 hr (step 4 ). The microspheres were then subjected to flow cytometric analysis (step 5 ). Minimums of 100 of each type of microsphere were read and the mean value of MESF was used for determining the genotypes. The fluorescence signal of the corresponding microsphere without SBCE reactions (microsphere alone) or SBCE reactions without AmpliTaq FS were subtracted from the MESF values.

    Article Snippet: Shrimp alkaline phosphatase (SAP) and Exonuclease I (Exo I) were obtained from Amersham Pharmacia.

    Techniques: Amplification, Polymerase Chain Reaction, Sequencing, Labeling, Luciferase, Hybridization, Flow Cytometry, Fluorescence

    Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) DNA sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) GFP expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.

    Journal: Journal of Virology

    Article Title: The DNase Activity of Kaposi’s Sarcoma-Associated Herpesvirus SOX Protein Serves an Important Role in Viral Genome Processing during Lytic Replication

    doi: 10.1128/JVI.01983-18

    Figure Lengend Snippet: Generation of ORF37-DNase-inactivated (Q129H) recombinant KSHV (the Q129H mutant) via homologous recombination. (A) Schematic diagram showing generation of the Q129H mutant, where amino acid residue 129 of ORF37 was point mutated to histidine by homologous recombination and GalK-Kan r counterselection. Insertion of the galK -Kan r cassette was confirmed by BamHI restriction fragment analysis and Southern hybridization with a GalK-specific probe, indicated in red (Probe). (B) EtBr-stained gel and Southern blot of BamHI-digested BAC16-wt (lane 2), an intermediate containing a galK -Kan r cassette (lane 3), and the Q129H mutant (lane 4). The Southern blot with a 32 P-labeled GalK probe showed the expected 2.6-kb band in the intermediate (lane 3). (C, a ) DNA sequencing of BAC Q129H confirmed the desired mutation in the ORF37 gene. ( b ) High-throughput sequencing reads of BAC Q129H confirmed mutation in the ORF37 gene. (D) GFP expression levels from HEK293L cells stably transfected with BAC16-wt or the Q129H DNAs post-hygromycin selection were imaged directly from the fixed cells by fluorescence microscopy. The green images show the expression of GFP, confirming the presence of BAC16-wt or the Q129H mutant, and the gray images are the corresponding bright-field images. (E) Comparison of expression levels of LANA, RTA, and ORF37 via Western blot analysis of uninduced and 48-hpi 293L/wt and 293L/Q129H cells confirmed the persistence and reactivation of both viruses. Staining of the same blot with GAPDH-specific antibody served as a loading control. (F, a ) Analysis of the host shutoff activity of the Q129H mutant using RNAs extracted from uninduced and 48-hpi 293L/wt and 293L/Q129H cells. RNA was resolved and subjected to Northern blotting and hybridization with a 32 P-labeled GFP probe, which confirmed that the Q129H mutant retained its ability to degrade the host mRNA. ( b ) Immunofluorescence analysis for RTA expression in 293L/wt and 293L/Q129H cells indicated almost all the cells underwent lytic reaction at 48 hpi. DIC, differential interference contrast. (G) Relative quantification of the viral mRNA levels from 48-hpi 293L/Q129H cells compared to the 293L/wt control using a custom real-time qPCR analysis of the KSHV genes in a 96-well format showed almost identical levels of all viral transcripts. The fold changes for the individual KSHV genes were calculated using the ΔΔ C T method normalized to GAPDH and relative to BAC16-wt, defined as 1.

    Article Snippet: Two micrograms of total RNA was resolved on a 1% agarose-formaldehyde gel (80 V for 3 h at 4°C), transferred to a Hybond-N+ membrane (GE Healthcare) using 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) transfer buffer, and hybridized with a 32 P-labeled DNA (GFP) probe generated using a random-primer DNA-labeling kit (Clontech Laboratories, Inc.).

    Techniques: Recombinant, Mutagenesis, Homologous Recombination, Hybridization, Staining, Southern Blot, Labeling, DNA Sequencing, BAC Assay, Next-Generation Sequencing, Expressing, Stable Transfection, Transfection, Selection, Fluorescence, Microscopy, Western Blot, Activity Assay, Northern Blot, Immunofluorescence, Real-time Polymerase Chain Reaction

    DNase activity of ORF37 did not affect latent/lytic KSHV DNA synthesis but rather virion production. (A and B) Comparison of intracellular KSHV genome copies in latent/uninduced and 48-hpi 293L/wt and 293L/Q129H cells, as analyzed by qPCR. The values were normalized to GAPDH and calculated relative to BAC16-wt, defined as 1. Equal numbers of KSHV genome copies were maintained during the latent phase; however, only a slight decrease in KSHV genome copies was observed during the lytic phase of virus replication. (C) Detection of extracellular KSHV progeny virion production in the supernatants of 5-dpi 293L/wt and 293L/Q129H cells revealed a 10-fold decrease in virus production in 293L/Q129H supernatants compared to wt supernatants. (D and E) HEK293L cells infected with 293L/wt and 293L/Q129H virus supernatants were assayed by flow cytometry 2 days postinfection. The side scatter (SSC) versus forward scatter (FSC) plot was used to identify GFP-positive cells/signals. (F) Mean value of at least three independent experiments from the values determined in panel E. The error bars represent standard deviations of the mean from the results of three different experiments; ***, P

    Journal: Journal of Virology

    Article Title: The DNase Activity of Kaposi’s Sarcoma-Associated Herpesvirus SOX Protein Serves an Important Role in Viral Genome Processing during Lytic Replication

    doi: 10.1128/JVI.01983-18

    Figure Lengend Snippet: DNase activity of ORF37 did not affect latent/lytic KSHV DNA synthesis but rather virion production. (A and B) Comparison of intracellular KSHV genome copies in latent/uninduced and 48-hpi 293L/wt and 293L/Q129H cells, as analyzed by qPCR. The values were normalized to GAPDH and calculated relative to BAC16-wt, defined as 1. Equal numbers of KSHV genome copies were maintained during the latent phase; however, only a slight decrease in KSHV genome copies was observed during the lytic phase of virus replication. (C) Detection of extracellular KSHV progeny virion production in the supernatants of 5-dpi 293L/wt and 293L/Q129H cells revealed a 10-fold decrease in virus production in 293L/Q129H supernatants compared to wt supernatants. (D and E) HEK293L cells infected with 293L/wt and 293L/Q129H virus supernatants were assayed by flow cytometry 2 days postinfection. The side scatter (SSC) versus forward scatter (FSC) plot was used to identify GFP-positive cells/signals. (F) Mean value of at least three independent experiments from the values determined in panel E. The error bars represent standard deviations of the mean from the results of three different experiments; ***, P

    Article Snippet: Two micrograms of total RNA was resolved on a 1% agarose-formaldehyde gel (80 V for 3 h at 4°C), transferred to a Hybond-N+ membrane (GE Healthcare) using 10× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) transfer buffer, and hybridized with a 32 P-labeled DNA (GFP) probe generated using a random-primer DNA-labeling kit (Clontech Laboratories, Inc.).

    Techniques: Activity Assay, DNA Synthesis, Real-time Polymerase Chain Reaction, Infection, Flow Cytometry, Cytometry