proteinase k Millipore Search Results


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  • 99
    Thermo Fisher proteinase k
    Treatment of needle proteins with proteinase K, lipoprotein lipase, and polymyxin B retains NF-κB/AP-1 activation. Needle proteins (1 μg/ml) (A, B, and C) and LcrG (1 μg/ml) (B and C), PBS, flagellin (1 μg/ml) (A), Pam3
    Proteinase K, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 16903 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore l protease k solution
    Treatment of needle proteins with proteinase K, lipoprotein lipase, and polymyxin B retains NF-κB/AP-1 activation. Needle proteins (1 μg/ml) (A, B, and C) and LcrG (1 μg/ml) (B and C), PBS, flagellin (1 μg/ml) (A), Pam3
    L Protease K Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore proteinase k
    The factor(s) in SP responsible for TrkA activation is degraded by <t>proteinase</t> K and has a molecular weight of 30–100 kDa. (A) Seminal plasma was incubated in the presence or absence of 200 μg/ml proteinase K for 18 h at 37°C, and then spiked with PMSF to inhibit further proteolytic activity. A final concentration of 1% or 10% SP (control, mock-treated, or proteinase-K treated) was exposed for 5 min to eSF from a woman without uterine pathologies, and then probed for pTrkA pY785 levels. (B) Seminal plasma was centrifuged using filter units with nominal molecular weight limits of 3, 10, 30, and 100 kDa to generate size fractions
    Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20789 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Merck KGaA proteinase k
    Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.
    Proteinase K, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 99/100, based on 717 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore proteinase k buffer pkb
    Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.
    Proteinase K Buffer Pkb, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Merck KGaA recombinant proteinase k
    Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.
    Recombinant Proteinase K, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 94/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore nuclease free proteinase k
    Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.
    Nuclease Free Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore proteinase k agarose
    In vivo  activation of DCs after U-Omp19 injection. U-Omp19 either untreated or digested with proteinase K,  E. coli  LPS or PBS alone was injected i.v. to BALB/c mice. Splenic CD11c +  DCs were analyzed for their activation status 20 h after injection by assessing the surface expression of ( A ) CD40, ( B ) CD80 and ( C ) CD86 molecules by flow cytometry. This experiment was conducted three times with similar results. Histograms display results from one representative experiment.
    Proteinase K Agarose, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore am 9624 proteinase k
    In vivo  activation of DCs after U-Omp19 injection. U-Omp19 either untreated or digested with proteinase K,  E. coli  LPS or PBS alone was injected i.v. to BALB/c mice. Splenic CD11c +  DCs were analyzed for their activation status 20 h after injection by assessing the surface expression of ( A ) CD40, ( B ) CD80 and ( C ) CD86 molecules by flow cytometry. This experiment was conducted three times with similar results. Histograms display results from one representative experiment.
    Am 9624 Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore rnase free proteinase k
    In vivo  activation of DCs after U-Omp19 injection. U-Omp19 either untreated or digested with proteinase K,  E. coli  LPS or PBS alone was injected i.v. to BALB/c mice. Splenic CD11c +  DCs were analyzed for their activation status 20 h after injection by assessing the surface expression of ( A ) CD40, ( B ) CD80 and ( C ) CD86 molecules by flow cytometry. This experiment was conducted three times with similar results. Histograms display results from one representative experiment.
    Rnase Free Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore dnase free proteinase k
    In vivo  activation of DCs after U-Omp19 injection. U-Omp19 either untreated or digested with proteinase K,  E. coli  LPS or PBS alone was injected i.v. to BALB/c mice. Splenic CD11c +  DCs were analyzed for their activation status 20 h after injection by assessing the surface expression of ( A ) CD40, ( B ) CD80 and ( C ) CD86 molecules by flow cytometry. This experiment was conducted three times with similar results. Histograms display results from one representative experiment.
    Dnase Free Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore proteinase k agarose beads
    Detection of CDK2 in HBV capsids purified from HepG2 cells by Western blotting. HBV capsids (0.5 μg) were digested with <t>proteinase</t> K (PK) agarose beads as described in Materials and Methods. Proteinase K was then inactivated by the addition of the proteinase K inhibitor, and the sample was resolved by SDS-PAGE (lanes 8 and 10), along with the same amount of undigested capsids from the same fractions (lanes 7 and 9). Purified GST-CDK2 standards (lanes 1 to 3) and total lysate from HepG2 cells (lane 4) were loaded as controls for CDK2 detection. A sucrose fraction that did not contain HBV capsids was also treated with the proteinase K agarose beads to ensure that contaminating proteins were removed by the digestion (lane 6) or mock treated (lane 5). The capsids loaded in lanes 9 and 10 were from a capsid-positive sucrose fraction of higher purity than those loaded in lanes 7 and 8. CDK2 was detected by Western blotting using the anti-CDK2 antibody (top panel). HBc was visualized by Sypro ruby staining (bottom panel). C, core protein.
    Proteinase K Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher proteinase k solution
    Detection of CDK2 in HBV capsids purified from HepG2 cells by Western blotting. HBV capsids (0.5 μg) were digested with <t>proteinase</t> K (PK) agarose beads as described in Materials and Methods. Proteinase K was then inactivated by the addition of the proteinase K inhibitor, and the sample was resolved by SDS-PAGE (lanes 8 and 10), along with the same amount of undigested capsids from the same fractions (lanes 7 and 9). Purified GST-CDK2 standards (lanes 1 to 3) and total lysate from HepG2 cells (lane 4) were loaded as controls for CDK2 detection. A sucrose fraction that did not contain HBV capsids was also treated with the proteinase K agarose beads to ensure that contaminating proteins were removed by the digestion (lane 6) or mock treated (lane 5). The capsids loaded in lanes 9 and 10 were from a capsid-positive sucrose fraction of higher purity than those loaded in lanes 7 and 8. CDK2 was detected by Western blotting using the anti-CDK2 antibody (top panel). HBc was visualized by Sypro ruby staining (bottom panel). C, core protein.
    Proteinase K Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Treatment of needle proteins with proteinase K, lipoprotein lipase, and polymyxin B retains NF-κB/AP-1 activation. Needle proteins (1 μg/ml) (A, B, and C) and LcrG (1 μg/ml) (B and C), PBS, flagellin (1 μg/ml) (A), Pam3

    Journal: Infection and Immunity

    Article Title: Type III Secretion Needle Proteins Induce Cell Signaling and Cytokine Secretion via Toll-Like Receptors

    doi: 10.1128/IAI.01705-14

    Figure Lengend Snippet: Treatment of needle proteins with proteinase K, lipoprotein lipase, and polymyxin B retains NF-κB/AP-1 activation. Needle proteins (1 μg/ml) (A, B, and C) and LcrG (1 μg/ml) (B and C), PBS, flagellin (1 μg/ml) (A), Pam3

    Article Snippet: Needle proteins and flagellin (standard flagellin from Salmonella Typhimurium; Invivogen) were incubated with 40 μg/ml proteinase K (Thermo-Fisher) at 37°C for 16 h. Proteinase K was then inactivated with 1.6 mg/ml phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich, St. Louis, MO).

    Techniques: Activation Assay

    The factor(s) in SP responsible for TrkA activation is degraded by proteinase K and has a molecular weight of 30–100 kDa. (A) Seminal plasma was incubated in the presence or absence of 200 μg/ml proteinase K for 18 h at 37°C, and then spiked with PMSF to inhibit further proteolytic activity. A final concentration of 1% or 10% SP (control, mock-treated, or proteinase-K treated) was exposed for 5 min to eSF from a woman without uterine pathologies, and then probed for pTrkA pY785 levels. (B) Seminal plasma was centrifuged using filter units with nominal molecular weight limits of 3, 10, 30, and 100 kDa to generate size fractions

    Journal: Biology of Reproduction

    Article Title: Potent and rapid activation of tropomyosin-receptor kinase A in endometrial stromal fibroblasts by seminal plasma

    doi: 10.1093/biolre/ioy056

    Figure Lengend Snippet: The factor(s) in SP responsible for TrkA activation is degraded by proteinase K and has a molecular weight of 30–100 kDa. (A) Seminal plasma was incubated in the presence or absence of 200 μg/ml proteinase K for 18 h at 37°C, and then spiked with PMSF to inhibit further proteolytic activity. A final concentration of 1% or 10% SP (control, mock-treated, or proteinase-K treated) was exposed for 5 min to eSF from a woman without uterine pathologies, and then probed for pTrkA pY785 levels. (B) Seminal plasma was centrifuged using filter units with nominal molecular weight limits of 3, 10, 30, and 100 kDa to generate size fractions

    Article Snippet: To confirm the degradation of seminal proteins by proteinase K, 200 μg/ml of proteinase K (Sigma-Aldrich) was added to 100% SP for 18 h at 37°C.

    Techniques: Activation Assay, Molecular Weight, Incubation, Activity Assay, Concentration Assay

    Western blot analysis of protein-resistant core (PrP res ) of pathological dromedary prion protein. A) Western blot analysis of proteinase K (PK)–treated PrP Sc in brain homogenates from dromedary camels with neurologic symptoms (nos. 4 and 8), Algeria. A sample of sheep scrapie was loaded as control (indicated as C+). Membranes were probed with L42 (left) and 12B2 monoclonal antibody (mAb) (right). Molecular weights (kDa) are indicated on the left. Tissue equivalents loaded per lane were 2 mg for camel samples and 0.1 mg for sheep scrapie. B) Samples after deglycosylation. Membrane was probed with L42 mAb. C) Comparison of dromedary PrP res (from camel no. 4) with sheep bovine spongiform encephalopathy (BSE), bovine BSE, and sheep scrapie samples by ISS (Istituto Superiore di Sanità) discriminatory Western blot ( 17 ). Tissue equivalents loaded per lane were 2 mg for dromedary camel and bovine samples and 0.1 mg for sheep samples. In each blot, samples were loaded as follows: lane 1, ovine BSE; lane 2, bovine BSE; lane 3, dromedary camel no. 4; lane 4, ovine scrapie. Membranes were probed with L42, 12B2, and SAF32 mAbs, as indicated. For the analyses in panels B and C, protein standards were loaded and are indicated as M.

    Journal: Emerging Infectious Diseases

    Article Title: Prion Disease in Dromedary Camels, Algeria

    doi: 10.3201/eid2406.172007

    Figure Lengend Snippet: Western blot analysis of protein-resistant core (PrP res ) of pathological dromedary prion protein. A) Western blot analysis of proteinase K (PK)–treated PrP Sc in brain homogenates from dromedary camels with neurologic symptoms (nos. 4 and 8), Algeria. A sample of sheep scrapie was loaded as control (indicated as C+). Membranes were probed with L42 (left) and 12B2 monoclonal antibody (mAb) (right). Molecular weights (kDa) are indicated on the left. Tissue equivalents loaded per lane were 2 mg for camel samples and 0.1 mg for sheep scrapie. B) Samples after deglycosylation. Membrane was probed with L42 mAb. C) Comparison of dromedary PrP res (from camel no. 4) with sheep bovine spongiform encephalopathy (BSE), bovine BSE, and sheep scrapie samples by ISS (Istituto Superiore di Sanità) discriminatory Western blot ( 17 ). Tissue equivalents loaded per lane were 2 mg for dromedary camel and bovine samples and 0.1 mg for sheep samples. In each blot, samples were loaded as follows: lane 1, ovine BSE; lane 2, bovine BSE; lane 3, dromedary camel no. 4; lane 4, ovine scrapie. Membranes were probed with L42, 12B2, and SAF32 mAbs, as indicated. For the analyses in panels B and C, protein standards were loaded and are indicated as M.

    Article Snippet: We performed membrane treatments, proteinase K (PK) (Sigma-Aldrich) digestion (50 μg/mL), and immunodetection as described ( ).

    Techniques: Western Blot

    SDS-PAGE of S. aureus cell wall extracts. (A) Clinical and laboratory strains grown in iron-rich (lanes 1, 3, and 5) and iron-restricted (lanes 2, 4, and 6) CRPMI showing major iron-regulated proteins of 40 and 87 kDa. Lanes: 1 and 2, BB; 3 and 4, 8325-4; 5 and 6, RN6390-B. (B) RN6390-B grown in iron-rich and iron-restricted CRPMI treated with proteinase K showing the surface exposure of the 40- and 87-kDa proteins. Lanes: 1, CRPMI; 2, CRPMI plus proteinase K; 3, iron-rich CRPMI; 4, iron-rich CRPMI plus proteinase K. (C) Fresh clinical isolates grown in CRPMI showing conservation of the 40- and 87-kDa proteins and demonstrating the size variation of both proteins. (D) Differential expression of 40- and 87-kDa proteins. Lanes: 1, RN6390-B recovered from chambers without subculture in vitro; 2 to 5, RN6390-B grown in CRPMI or TSM (lanes 2 and 4) or iron-rich CRPMI or TSM (lanes 3 and 5). (E) Immunoblot of RN6390-B cell wall extracts with pooled human serum. Lanes: 1, cells grown in vivo; 2, CRPMI; 3, iron-rich CRPMI; 4, TSM; 5, iron-rich TSM; 6, LB. The 87- and 40-kDa proteins are indicated. M, size marker.

    Journal: Infection and Immunity

    Article Title: Conservation, Surface Exposure, and In Vivo Expression of the Frp Family of Iron-Regulated Cell Wall Proteins in Staphylococcus aureus

    doi: 10.1128/IAI.70.5.2399-2407.2002

    Figure Lengend Snippet: SDS-PAGE of S. aureus cell wall extracts. (A) Clinical and laboratory strains grown in iron-rich (lanes 1, 3, and 5) and iron-restricted (lanes 2, 4, and 6) CRPMI showing major iron-regulated proteins of 40 and 87 kDa. Lanes: 1 and 2, BB; 3 and 4, 8325-4; 5 and 6, RN6390-B. (B) RN6390-B grown in iron-rich and iron-restricted CRPMI treated with proteinase K showing the surface exposure of the 40- and 87-kDa proteins. Lanes: 1, CRPMI; 2, CRPMI plus proteinase K; 3, iron-rich CRPMI; 4, iron-rich CRPMI plus proteinase K. (C) Fresh clinical isolates grown in CRPMI showing conservation of the 40- and 87-kDa proteins and demonstrating the size variation of both proteins. (D) Differential expression of 40- and 87-kDa proteins. Lanes: 1, RN6390-B recovered from chambers without subculture in vitro; 2 to 5, RN6390-B grown in CRPMI or TSM (lanes 2 and 4) or iron-rich CRPMI or TSM (lanes 3 and 5). (E) Immunoblot of RN6390-B cell wall extracts with pooled human serum. Lanes: 1, cells grown in vivo; 2, CRPMI; 3, iron-rich CRPMI; 4, TSM; 5, iron-rich TSM; 6, LB. The 87- and 40-kDa proteins are indicated. M, size marker.

    Article Snippet: Proteinase K (Boehringer Mannheim) was added to 1-ml aliquots of S. aureus suspension to a final concentration of 0.1 mg/ml, and control and proteinase K-treated samples were incubated at 37°C for 1 h. Bacteria were pelleted and washed four times in PBS plus 1 mM phenylmethylsulfonyl fluoride (Sigma).

    Techniques: SDS Page, Expressing, In Vitro, In Vivo, Marker

    The lactobacilli-CFS high Mw fraction contains an isolated heat sensitive lipid-based factor. Molecular characterization of the IFN-γ dampening factor of the Lactobacillus -CFS high Mw fraction. The Lactobacillus -CFS high Mw fraction was subjected to high performance liquid chromatography (HPLC), proteinase K treatment, heat inactivation or delipidation through lipid adsorption before being added to S. aureus (S.a)-CFS stimulated PBMC. ( a ) The LGG-CFS was spin-column fractionated with a pore-size of 100 kDa, after which the top fraction was collected and subjected to further size fractionation using HPLC. The HPLC generated fractions were added to S.a-CFS stimulated PBMC for 48 h followed by quantification of secreted levels of IFN-γ normalized to S.a-CFS alone, (n = 8). ( b ) PBMC were stimulated 48 h in the presence or absence of proteinase K treated, heat treated and control Lactobacillus -CFS whereby relative secreted levels of IFN-γ was analysed, (n = 8). ( c ) PBMC were stimulated 48 h in the presence or absence of delipidated or control Lactobacillus -CFS whereby relative secreted levels of IFN-γ was analysed (n = 8). Shown are medians with interquartile range.

    Journal: Scientific Reports

    Article Title: Extracellular Membrane Vesicles from Lactobacilli Dampen IFN-γ Responses in a Monocyte-Dependent Manner

    doi: 10.1038/s41598-019-53576-6

    Figure Lengend Snippet: The lactobacilli-CFS high Mw fraction contains an isolated heat sensitive lipid-based factor. Molecular characterization of the IFN-γ dampening factor of the Lactobacillus -CFS high Mw fraction. The Lactobacillus -CFS high Mw fraction was subjected to high performance liquid chromatography (HPLC), proteinase K treatment, heat inactivation or delipidation through lipid adsorption before being added to S. aureus (S.a)-CFS stimulated PBMC. ( a ) The LGG-CFS was spin-column fractionated with a pore-size of 100 kDa, after which the top fraction was collected and subjected to further size fractionation using HPLC. The HPLC generated fractions were added to S.a-CFS stimulated PBMC for 48 h followed by quantification of secreted levels of IFN-γ normalized to S.a-CFS alone, (n = 8). ( b ) PBMC were stimulated 48 h in the presence or absence of proteinase K treated, heat treated and control Lactobacillus -CFS whereby relative secreted levels of IFN-γ was analysed, (n = 8). ( c ) PBMC were stimulated 48 h in the presence or absence of delipidated or control Lactobacillus -CFS whereby relative secreted levels of IFN-γ was analysed (n = 8). Shown are medians with interquartile range.

    Article Snippet: Proteinase K digestion and heat treatment The Lactobacillus -CFS was subjected to protein digestion using Proteinase K immobilized to agarose (Sigma-Aldrich) using a modified protocol of that described elsewhere .

    Techniques: Isolation, High Performance Liquid Chromatography, Adsorption, Fractionation, Generated

    Proteins in TI 1068 LWW Inhibit P. tabacina Spore Germination and Leaf Infection. (A) P. tabacina spore germination assay (Pt), Coomassie blue–stained SDS-PAGE gel blot (sds), and protein gel blot with a 1:10,000 dilution of phylloplanin antiserum (w). Gel a, water plus spores; gel b, TI 1068 LWW (diluted to 100 ng/μL total protein) plus spores; gel c, TI 1068 LWW (100 ng/μL total protein) digested with proteinase K (ProtK) plus spores. The arrow marks residual, soluble proteinase K. (B) P. tabacina leaf infection assay of cv Petite Havana. Photograph a, water plus spores (10 4 spores/mL); a sporulating lesion is indicated with the arrow. Photograph b, TI 1068 LWW (diluted to 50 ng/μL) plus spores (10 4 spores/mL).

    Journal: The Plant Cell

    Article Title: Phylloplanins of Tobacco Are Defensive Proteins Deployed on Aerial Surfaces by Short Glandular Trichomes

    doi: 10.1105/tpc.105.031559

    Figure Lengend Snippet: Proteins in TI 1068 LWW Inhibit P. tabacina Spore Germination and Leaf Infection. (A) P. tabacina spore germination assay (Pt), Coomassie blue–stained SDS-PAGE gel blot (sds), and protein gel blot with a 1:10,000 dilution of phylloplanin antiserum (w). Gel a, water plus spores; gel b, TI 1068 LWW (diluted to 100 ng/μL total protein) plus spores; gel c, TI 1068 LWW (100 ng/μL total protein) digested with proteinase K (ProtK) plus spores. The arrow marks residual, soluble proteinase K. (B) P. tabacina leaf infection assay of cv Petite Havana. Photograph a, water plus spores (10 4 spores/mL); a sporulating lesion is indicated with the arrow. Photograph b, TI 1068 LWW (diluted to 50 ng/μL) plus spores (10 4 spores/mL).

    Article Snippet: Insoluble proteinase K affixed to acrylic beads (100 mg; P0803; Sigma-Aldrich) was placed into mini-spin filters (732-6027; Bio-Rad).

    Techniques: Infection, Germination Assay, Staining, SDS Page, Western Blot

    E. coli –Expressed TI 1068 T-Phylloplanin Inhibits P. tabacina Spore Germination. Coomassie blue–stained SDS-PAGE gel blots (sds), protein gel blots with a 1:10,000 dilution of T-phylloplanin antiserum (w), and P. tabacina spore germination assays (Pt). (A) E. coli –expressed MBP-PhyllP (160 ng/μL total protein) treated with factor Xa. The arrow indicates released T-PhyllP. (B) E. coli –expressed MBP-T-PhyllP (160 ng/μL total protein) treated with factor Xa and proteinase-K (ProtK). The volume used was equivalent to that in (A) . (C) E. coli –expressed MBP (200 ng/μL total protein) treated with factor Xa. (D) E. coli –expressed MBP (200 ng/μL total protein) treated with factor Xa and proteinase K. The volume used was equivalent to that in (C) .

    Journal: The Plant Cell

    Article Title: Phylloplanins of Tobacco Are Defensive Proteins Deployed on Aerial Surfaces by Short Glandular Trichomes

    doi: 10.1105/tpc.105.031559

    Figure Lengend Snippet: E. coli –Expressed TI 1068 T-Phylloplanin Inhibits P. tabacina Spore Germination. Coomassie blue–stained SDS-PAGE gel blots (sds), protein gel blots with a 1:10,000 dilution of T-phylloplanin antiserum (w), and P. tabacina spore germination assays (Pt). (A) E. coli –expressed MBP-PhyllP (160 ng/μL total protein) treated with factor Xa. The arrow indicates released T-PhyllP. (B) E. coli –expressed MBP-T-PhyllP (160 ng/μL total protein) treated with factor Xa and proteinase-K (ProtK). The volume used was equivalent to that in (A) . (C) E. coli –expressed MBP (200 ng/μL total protein) treated with factor Xa. (D) E. coli –expressed MBP (200 ng/μL total protein) treated with factor Xa and proteinase K. The volume used was equivalent to that in (C) .

    Article Snippet: Insoluble proteinase K affixed to acrylic beads (100 mg; P0803; Sigma-Aldrich) was placed into mini-spin filters (732-6027; Bio-Rad).

    Techniques: Staining, SDS Page

    Changes in the unit-cell volume in initial crystal characterization. During initial characterization the unit-cell volume change was monitored with respect to the initial 295 K unit cell. The unit-cell volume decrease upon cryoprotection is shown in yellow, the unit-cell volume decrease upon cryocooling naked crystals is presented in purple and the unit-cell volume decrease upon cryocooling naked crystals at a RH of 91% is shown in green. Each set of bars represents one protein crystal system: 1, BAZ2B; 2, Tudor; 3, JMJD2D; 4, MINA53; 5, PHIP(2); 6, TPH2; 7, proteinase K; 8, glucose isomerase ( I 222). PHIP(2) did not tolerate dehydration, so there is no result for this system.  P 222 glucose isomerase did not occur at 295 K so only  I 222 glucose isomerase is represented.

    Journal: Acta Crystallographica. Section D, Structural Biology

    Article Title: A generic protocol for protein crystal dehydration using the HC1b humidity controller

    doi: 10.1107/S2059798316003065

    Figure Lengend Snippet: Changes in the unit-cell volume in initial crystal characterization. During initial characterization the unit-cell volume change was monitored with respect to the initial 295 K unit cell. The unit-cell volume decrease upon cryoprotection is shown in yellow, the unit-cell volume decrease upon cryocooling naked crystals is presented in purple and the unit-cell volume decrease upon cryocooling naked crystals at a RH of 91% is shown in green. Each set of bars represents one protein crystal system: 1, BAZ2B; 2, Tudor; 3, JMJD2D; 4, MINA53; 5, PHIP(2); 6, TPH2; 7, proteinase K; 8, glucose isomerase ( I 222). PHIP(2) did not tolerate dehydration, so there is no result for this system. P 222 glucose isomerase did not occur at 295 K so only I 222 glucose isomerase is represented.

    Article Snippet: Plate-like crystals (100 × 80 × 20 µm) appeared overnight from sitting-drop plates at 293 K. Proteinase K from Tritirachium album (Sigma–Aldrich, catalogue No. P2308) was crystallized by mixing 1 µl 25 mg ml−1 protein solution in 25 mM HEPES pH 7.0, 100 µM PMSF with 1 µl reservoir solution consisting of 0.1 M bis-tris pH 5.5, 0.65 M LiCl.

    Techniques:

    Effect on splenocyte survival of diverse pretreatments applied to the non-cytosolic trypanosome fraction (NCF). Splenocytes were exposed for 75 min to the non-cytosolic fraction corresponding to 4 10 8 trypanosome equivalents per ml. Mock, PBS; NCF 37°C and 4°C, non-cytosolic fraction kept at 37 or 4°C respectively for 24 h prior to incorporation onto the cells; Pf, E64 and PK, treatment of NCF with Pefablock or E64 or proteinase K respectively for 24 hours prior to incorporation onto cells. The three cell subsets represented are normocytic and annexinV/PI double negative (red, healthy), normocytic and annexinV/PI double positive (green, early necrosis), and microcytic and annexinV/PI double positive (blue, late necrosis). The values presented are means of three independent experiments.

    Journal: PLoS ONE

    Article Title: A Non-Cytosolic Protein of Trypanosoma evansi Induces CD45-Dependent Lymphocyte Death

    doi: 10.1371/journal.pone.0005728

    Figure Lengend Snippet: Effect on splenocyte survival of diverse pretreatments applied to the non-cytosolic trypanosome fraction (NCF). Splenocytes were exposed for 75 min to the non-cytosolic fraction corresponding to 4 10 8 trypanosome equivalents per ml. Mock, PBS; NCF 37°C and 4°C, non-cytosolic fraction kept at 37 or 4°C respectively for 24 h prior to incorporation onto the cells; Pf, E64 and PK, treatment of NCF with Pefablock or E64 or proteinase K respectively for 24 hours prior to incorporation onto cells. The three cell subsets represented are normocytic and annexinV/PI double negative (red, healthy), normocytic and annexinV/PI double positive (green, early necrosis), and microcytic and annexinV/PI double positive (blue, late necrosis). The values presented are means of three independent experiments.

    Article Snippet: Different conditions were imposed during storage: (i) 4 or 37°C, (ii) 4 or 37°C with/without inhibitors of serine proteases (Pefabloc® SC, 1 mM, Fluka), (iii) 37°C with/without inhibition of cysteine proteases (E64, 10 µM, Sigma-Aldrich), (iv) 4 or 37°C with/without proteinase K (5 U/ml, Sigma-Aldrich), and (v) 37°C with/without DNAse I (2000 U/ml, Invitrogen).

    Techniques:

    Proteinase-K treated HPSE results in a reduced cytokine response in PBMCs. HPSE (1 µg) was treated with proteinase-K agarose beads (80 µg/ml) for 30 min at 37°C before removal of proteinase-K agarose beads and SDS-PAGE analysis. ( A ) Western blot analysis for HPSE confirms that proteinase-K treatment dramatically reduced intact HPSE levels in the sample. ( B ) HPSE activity assay on identically treated samples of either HPSE alone, proteinase K alone, or HPSE treated with proteinase K. Data represent the mean ±SEM (n = 3). ( C ) Expression of cytokines after stimulation with proteinase-K treated HPSE in IL-1β, IL-6, IL-10, IL-8 and TNF in PBMCs isolated from human whole blood. Data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p =

    Journal: PLoS ONE

    Article Title: Soluble Heparan Sulfate Fragments Generated by Heparanase Trigger the Release of Pro-Inflammatory Cytokines through TLR-4

    doi: 10.1371/journal.pone.0109596

    Figure Lengend Snippet: Proteinase-K treated HPSE results in a reduced cytokine response in PBMCs. HPSE (1 µg) was treated with proteinase-K agarose beads (80 µg/ml) for 30 min at 37°C before removal of proteinase-K agarose beads and SDS-PAGE analysis. ( A ) Western blot analysis for HPSE confirms that proteinase-K treatment dramatically reduced intact HPSE levels in the sample. ( B ) HPSE activity assay on identically treated samples of either HPSE alone, proteinase K alone, or HPSE treated with proteinase K. Data represent the mean ±SEM (n = 3). ( C ) Expression of cytokines after stimulation with proteinase-K treated HPSE in IL-1β, IL-6, IL-10, IL-8 and TNF in PBMCs isolated from human whole blood. Data represent the mean ±SEM of triplicate samples, results are representative of three independent experiments. *p =

    Article Snippet: Proteinase-K treatment of HPSE Proteinase-K-agarose beads (Sigma Aldrich) were suspended in endotoxin free PBS (Ca2+ - and Mg2+ -free, Life Technologies, CA) to a concentration of 80 mg/ml.

    Techniques: SDS Page, Western Blot, Activity Assay, Expressing, Isolation

    Alpha-synuclein immunohistochemistry after proteinase K treatment in L444P/+ and KO/+ mouse brains. ( A – C ) Accumulation of small alpha-synuclein proteinase K-resistant aggregates in the polymorph layer of the hippocampal dendate gyrus ( A ), stratum lucidum of the hippocampal cornu ammonis 3 (CA3) region ( B ) and striatum ( C ) of KO/+ mice, but not L444P/+ mice compared to their corresponding wild-type ( +/+ ) control littermates. ( D – E ) No proteinase K-resistant alpha-synuclein aggregates were found in the substantia nigra ( D ) and parvocellular reticular nucleus of the brainstem ( E ) of KO/+ and L444P/+ mice compared to their corresponding wild-type control littermates. Scale bars = 100 μm. Representative images are shown. Three mice of each genotype were analysed.

    Journal: Brain

    Article Title: The L444P Gba1 mutation enhances alpha-synuclein induced loss of nigral dopaminergic neurons in mice

    doi: 10.1093/brain/awx221

    Figure Lengend Snippet: Alpha-synuclein immunohistochemistry after proteinase K treatment in L444P/+ and KO/+ mouse brains. ( A – C ) Accumulation of small alpha-synuclein proteinase K-resistant aggregates in the polymorph layer of the hippocampal dendate gyrus ( A ), stratum lucidum of the hippocampal cornu ammonis 3 (CA3) region ( B ) and striatum ( C ) of KO/+ mice, but not L444P/+ mice compared to their corresponding wild-type ( +/+ ) control littermates. ( D – E ) No proteinase K-resistant alpha-synuclein aggregates were found in the substantia nigra ( D ) and parvocellular reticular nucleus of the brainstem ( E ) of KO/+ and L444P/+ mice compared to their corresponding wild-type control littermates. Scale bars = 100 μm. Representative images are shown. Three mice of each genotype were analysed.

    Article Snippet: Antigen retrieval was performed for alpha-synuclein and ionized calcium binding adaptor molecule 1 (Iba1) staining, by incubating the sections in 10 mM sodium citrate (Sigma-Aldrich), pH 8.5, at 80°C for 30 min; for antigen retrieval of glial fibrillary acidic protein (GFAP), sections were incubated in proteinase K solution (Sigma-Aldrich) in Tris-EDTA (TE) buffer (50 mM Tris base, 1 mM EDTA, 0.5% Triton™ X-100, pH 8.0), 20 µg/ml, at room temperature for 5 min. Proteinase K activity was blocked by incubation adding 5 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich).

    Techniques: Immunohistochemistry, Mouse Assay

    Induction of Jurkat cell apoptosis by MV is enhanced by, but not dependent on, carriage of VacA. 60190 MV were pretreated with proteinase K (PK) (inactivated using a protease inhibitor [PI] prior to cell culture). Treated and untreated MV were added to

    Journal: Infection and Immunity

    Article Title: Helicobacter pylori Membrane Vesicles Stimulate Innate Pro- and Anti-Inflammatory Responses and Induce Apoptosis in Jurkat T Cells

    doi: 10.1128/IAI.01443-13

    Figure Lengend Snippet: Induction of Jurkat cell apoptosis by MV is enhanced by, but not dependent on, carriage of VacA. 60190 MV were pretreated with proteinase K (PK) (inactivated using a protease inhibitor [PI] prior to cell culture). Treated and untreated MV were added to

    Article Snippet: Proteinase K-treated MV (and an equivalent buffer-only control) were preincubated with 100 μg/ml proteinase K for 60 min at 37°C following the methods of Bomberger et al. , and then proteinase K was inactivated using P1860 protease inhibitor cocktail (Sigma) before adding the MV to the T cells.

    Techniques: Protease Inhibitor, Cell Culture

    MicroED structure of proteinase K at 1.75 Å resolution. ( a ) The overall MicroED structure of proteinase K. ( b ) A five-residue fragment of the final model refined against the data derived from the corrected images. The SA composite omit map at 1.75 Å resolution is contoured at 1.0σ above the mean and shows a hole in the center of the tyrosine side chain. The figures were generated using PyMol (Schrödinger, 2014 ▸ ).

    Journal: Journal of Applied Crystallography

    Article Title: Modeling truncated pixel values of faint reflections in MicroED images 1

    doi: 10.1107/S1600576716007196

    Figure Lengend Snippet: MicroED structure of proteinase K at 1.75 Å resolution. ( a ) The overall MicroED structure of proteinase K. ( b ) A five-residue fragment of the final model refined against the data derived from the corrected images. The SA composite omit map at 1.75 Å resolution is contoured at 1.0σ above the mean and shows a hole in the center of the tyrosine side chain. The figures were generated using PyMol (Schrödinger, 2014 ▸ ).

    Article Snippet: The procedure was validated against MicroED images collected from four crystals of proteinase K. Protein solutions from Engyodontium album (Sigma-Aldrich, St Louis, MO, USA) were prepared by combining 2 µl of protein solution (50 mg ml−1 ) with 2 µl of precipitant solution (1.0–1.3 M ammonium sulfate, 0.1 M Tris pH 8.0).

    Techniques: Derivative Assay, Generated

    Correlation coefficients of the refined model. ( a ) Particularly at high resolution, CC work  (solid curves) and CC free  (dashed curves) are generally higher for the model refinement against the corrected dataset (orange curves) than for the model refined against the uncorrected dataset (black curves). ( b ) For all 279 residues of proteinase K, the real-space correlation coefficient for the corrected data in the resolution range between 1.75 and 5.00 Å is higher for the model refined against the corrected data than for the model refined against the same resolution range of the uncorrected data.

    Journal: Journal of Applied Crystallography

    Article Title: Modeling truncated pixel values of faint reflections in MicroED images 1

    doi: 10.1107/S1600576716007196

    Figure Lengend Snippet: Correlation coefficients of the refined model. ( a ) Particularly at high resolution, CC work (solid curves) and CC free (dashed curves) are generally higher for the model refinement against the corrected dataset (orange curves) than for the model refined against the uncorrected dataset (black curves). ( b ) For all 279 residues of proteinase K, the real-space correlation coefficient for the corrected data in the resolution range between 1.75 and 5.00 Å is higher for the model refined against the corrected data than for the model refined against the same resolution range of the uncorrected data.

    Article Snippet: The procedure was validated against MicroED images collected from four crystals of proteinase K. Protein solutions from Engyodontium album (Sigma-Aldrich, St Louis, MO, USA) were prepared by combining 2 µl of protein solution (50 mg ml−1 ) with 2 µl of precipitant solution (1.0–1.3 M ammonium sulfate, 0.1 M Tris pH 8.0).

    Techniques:

    Distribution of the low counts in a typical MicroED image of proteinase K collected by continuous rotation using the rolling shutter mode of the camera. ( a ) The histogram in the second outermost shell between 1.5 and 1.7 Å for an uncorrected image, and ( b ) the histogram of the corresponding corrected image. The continuous curves in ( b ) show the fitted lognormal distributions in the two innermost (resolutions lower than 4.7 Å in blue, resolutions between 3.4 and 4.7 Å in orange) and the second outermost (black curve) resolution shells. As the resolution increases, the mode and the variance of the distribution decrease.

    Journal: Journal of Applied Crystallography

    Article Title: Modeling truncated pixel values of faint reflections in MicroED images 1

    doi: 10.1107/S1600576716007196

    Figure Lengend Snippet: Distribution of the low counts in a typical MicroED image of proteinase K collected by continuous rotation using the rolling shutter mode of the camera. ( a ) The histogram in the second outermost shell between 1.5 and 1.7 Å for an uncorrected image, and ( b ) the histogram of the corresponding corrected image. The continuous curves in ( b ) show the fitted lognormal distributions in the two innermost (resolutions lower than 4.7 Å in blue, resolutions between 3.4 and 4.7 Å in orange) and the second outermost (black curve) resolution shells. As the resolution increases, the mode and the variance of the distribution decrease.

    Article Snippet: The procedure was validated against MicroED images collected from four crystals of proteinase K. Protein solutions from Engyodontium album (Sigma-Aldrich, St Louis, MO, USA) were prepared by combining 2 µl of protein solution (50 mg ml−1 ) with 2 µl of precipitant solution (1.0–1.3 M ammonium sulfate, 0.1 M Tris pH 8.0).

    Techniques:

    Fragment ion spectra of the Proteinase K generated plasminogen O -glycopeptide 362 LAP T APPELTPV 373 measured with nanoRP-LC-ESI MS n (positive ion mode, CID and ETD). A (top), For the given O -glycopeptide the CID-MS 2 spectrum is shown together with its corresponding precursor ion m / z 718.30 [M+3H] 3+ (inset). The spectrum allows the elucidation of the O -glycan composition (here disialylated T-antigen). In addition, also some internal glycopeptide fragments have been detected ( e.g. b 10 +HexNAc). A (bottom): The putative peptide mass ( m / z 1205.66 [M+H] + ) of the given O -glycopeptide was subjected to CID-MS 3 fragmentation. The peptide was identified by MASCOT search (Score: 16, UniProt KB/Swiss-Prot, human). B, The O -glycosylation site (here Thr 365 ) was pinpointed by means of ETD (Biotools-Score: 150). Magnified regions show the isotope pattern of selected peptide fragment ions, confirming the annotation. In addition to peptide fragment ions also fragment ions derived from the glycan moiety were detected, allowing a verification of the glycan composition. Furthermore, a neutral loss of an acetyl radical from the intact O -glycopeptide was observed, which is typically seen in ETD spectra of glycopeptides.

    Journal: Molecular & Cellular Proteomics : MCP

    Article Title: Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins *

    doi: 10.1074/mcp.M115.053546

    Figure Lengend Snippet: Fragment ion spectra of the Proteinase K generated plasminogen O -glycopeptide 362 LAP T APPELTPV 373 measured with nanoRP-LC-ESI MS n (positive ion mode, CID and ETD). A (top), For the given O -glycopeptide the CID-MS 2 spectrum is shown together with its corresponding precursor ion m / z 718.30 [M+3H] 3+ (inset). The spectrum allows the elucidation of the O -glycan composition (here disialylated T-antigen). In addition, also some internal glycopeptide fragments have been detected ( e.g. b 10 +HexNAc). A (bottom): The putative peptide mass ( m / z 1205.66 [M+H] + ) of the given O -glycopeptide was subjected to CID-MS 3 fragmentation. The peptide was identified by MASCOT search (Score: 16, UniProt KB/Swiss-Prot, human). B, The O -glycosylation site (here Thr 365 ) was pinpointed by means of ETD (Biotools-Score: 150). Magnified regions show the isotope pattern of selected peptide fragment ions, confirming the annotation. In addition to peptide fragment ions also fragment ions derived from the glycan moiety were detected, allowing a verification of the glycan composition. Furthermore, a neutral loss of an acetyl radical from the intact O -glycopeptide was observed, which is typically seen in ETD spectra of glycopeptides.

    Article Snippet: Proteinase K Digestion Proteolytic digestion was achieved by addition of Proteinase K (Sigma Aldrich), a serine protease with a broad specificity that cleaves primarily after aliphatic, aromatic and hydrophobic amino acids.

    Techniques: Generated, Mass Spectrometry, Derivative Assay

    Dot blot assay for aggregated human aSyn in A53T mice. The tissue samples were homogenized and incubated for 10 min with proteinase K to digest the soluble forms of aSyn. Samples were blotted and revealed through an antihuman aSyn antibody to visualize proteinase K-resistant fraction. Each row shows a scheme of the analyzed area, the quantification of n =5 blots and an exemplary blot for a . cortex; b . hippocampus; c . midbrain; and d . cerebellum

    Journal: Neuromolecular Medicine

    Article Title: Deferiprone Rescues Behavioral Deficits Induced by Mild Iron Exposure in a Mouse Model of Alpha-Synuclein Aggregation

    doi: 10.1007/s12017-017-8447-9

    Figure Lengend Snippet: Dot blot assay for aggregated human aSyn in A53T mice. The tissue samples were homogenized and incubated for 10 min with proteinase K to digest the soluble forms of aSyn. Samples were blotted and revealed through an antihuman aSyn antibody to visualize proteinase K-resistant fraction. Each row shows a scheme of the analyzed area, the quantification of n =5 blots and an exemplary blot for a . cortex; b . hippocampus; c . midbrain; and d . cerebellum

    Article Snippet: Dot blots were performed as previously described (Oprandy et al. ) in order to visualize all possible proteinase K-resistant forms of aSyn, by using a Minifold I system (Sigma, #WHA10447850).

    Techniques: Dot Blot, Mouse Assay, Incubation

    Comparison between GFP fluorescence, oxidative stress levels, cell viability and proteinase K (PK) resistance for Aβ42-GFP mutants after 16 and 48 h of expression. (A) Representative fluorescence microscopy images showing GFP signal of yeast cells expressing selected Aβ42-GFP variants (Gln, Thr, Ile and F19D/L34P) for 48 h. Scale bar represents 10 µm. (B) CellROX median fluorescence bar graph of yeast cultures expressing selected Aβ42-GFP variants induced for 16 and 48 h determined by FC analysis. Error bars represent CV from 10,000 events. (C) Percentage of IP positive cells in cultures expressing selected Aβ42-GFP variants induced for indicated times. Error bars represent ±SE (n=3). (D) Time-lapse PK resistance of insoluble fractions of selected Aβ42-GFP variants, induced for 16 and 48 h, detected by western blotting. Detected bands correspond to MW =35 kDa. Cells expressing GFP alone were used as control.

    Journal: Redox Biology

    Article Title: Protein aggregation into insoluble deposits protects from oxidative stress

    doi: 10.1016/j.redox.2017.03.027

    Figure Lengend Snippet: Comparison between GFP fluorescence, oxidative stress levels, cell viability and proteinase K (PK) resistance for Aβ42-GFP mutants after 16 and 48 h of expression. (A) Representative fluorescence microscopy images showing GFP signal of yeast cells expressing selected Aβ42-GFP variants (Gln, Thr, Ile and F19D/L34P) for 48 h. Scale bar represents 10 µm. (B) CellROX median fluorescence bar graph of yeast cultures expressing selected Aβ42-GFP variants induced for 16 and 48 h determined by FC analysis. Error bars represent CV from 10,000 events. (C) Percentage of IP positive cells in cultures expressing selected Aβ42-GFP variants induced for indicated times. Error bars represent ±SE (n=3). (D) Time-lapse PK resistance of insoluble fractions of selected Aβ42-GFP variants, induced for 16 and 48 h, detected by western blotting. Detected bands correspond to MW =35 kDa. Cells expressing GFP alone were used as control.

    Article Snippet: 2.11 Proteinase K resistance assay Insoluble fractions, obtained as described above, were incubated with 10 μg/mL proteinase K (PK) (Sigma-Aldrich) in PBS at 37 °C.

    Techniques: Fluorescence, Expressing, Microscopy, Western Blot

    Quantification of signals for phosphorylated CagA ( -CagA) and CagA. Infection times were chosen for 1 hour ( A , B ) or 3 hours ( C , D ). Densitometry analysis of the signals from the immunodetection of CagA and its phosphorylated form -CagA. Signals from traditional harvest by scrapping (Trad) were used as normalization parameter stabilizing the 100% of the signals to be obtained. Signals obtained from samples digested Trypsin EDTA and Proteinase K are shown relative to the traditional harvest. Single examples of the blots for each time point are shown as well ( B , D ). All densitometry values were normalized to the Stain Free signal.

    Journal: Scientific Reports

    Article Title: The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated

    doi: 10.1038/srep23227

    Figure Lengend Snippet: Quantification of signals for phosphorylated CagA ( -CagA) and CagA. Infection times were chosen for 1 hour ( A , B ) or 3 hours ( C , D ). Densitometry analysis of the signals from the immunodetection of CagA and its phosphorylated form -CagA. Signals from traditional harvest by scrapping (Trad) were used as normalization parameter stabilizing the 100% of the signals to be obtained. Signals obtained from samples digested Trypsin EDTA and Proteinase K are shown relative to the traditional harvest. Single examples of the blots for each time point are shown as well ( B , D ). All densitometry values were normalized to the Stain Free signal.

    Article Snippet: Three treatments were used for harvest: i) Traditional (Trad), in which the cells were placed 5 hours in 16 °C and collected using cell scrapers to remove them from the growth surface in 1 ml cold PBS containing 1 mM PMSF, 1 mM Sodium Ortho-vanadat, 1 μM Leupetin and 1 μM pepstatin. ii) Trypsin-EDTA (TE), in which cells were removed from the surface using 0.5 ml of Trypsin-EDTA solution (Gibco, Life Technologies) and incubated for 5 hours at 16 °C, at which time they had lost adherence to the growth surface and where collected. iii) Proteinase K digestion, which it was achieved using 1 ml per well of a 10 mg/ml crude Proteinase K (Sigma) in Proteinase K buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2 , 1 mM MgCl2 , 1 mM KCl) for 5 hours at 16 °C, after which cells were detached from the growth surface.

    Techniques: Infection, Immunodetection, Staining

    Establishment of different harvesting methods for the quantification of CagA injection. ( A ) Schematic representation of the experimental setup explained in Material and Methods section. ( B ) GFP expressing P12 strain (P12 GFP) infected AGS cells for one hour. Cells were harvested by scrapping (Traditional, Trad), Trypsin-EDTA (TE) or Proteinase K (PK) digestion. Mean fluorescence Units are shown here. Cells without infection are shown as control for changes in fluorescence caused by the different treatments. n = 3, ANOVA used for the statistical analysis. *P

    Journal: Scientific Reports

    Article Title: The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated

    doi: 10.1038/srep23227

    Figure Lengend Snippet: Establishment of different harvesting methods for the quantification of CagA injection. ( A ) Schematic representation of the experimental setup explained in Material and Methods section. ( B ) GFP expressing P12 strain (P12 GFP) infected AGS cells for one hour. Cells were harvested by scrapping (Traditional, Trad), Trypsin-EDTA (TE) or Proteinase K (PK) digestion. Mean fluorescence Units are shown here. Cells without infection are shown as control for changes in fluorescence caused by the different treatments. n = 3, ANOVA used for the statistical analysis. *P

    Article Snippet: Three treatments were used for harvest: i) Traditional (Trad), in which the cells were placed 5 hours in 16 °C and collected using cell scrapers to remove them from the growth surface in 1 ml cold PBS containing 1 mM PMSF, 1 mM Sodium Ortho-vanadat, 1 μM Leupetin and 1 μM pepstatin. ii) Trypsin-EDTA (TE), in which cells were removed from the surface using 0.5 ml of Trypsin-EDTA solution (Gibco, Life Technologies) and incubated for 5 hours at 16 °C, at which time they had lost adherence to the growth surface and where collected. iii) Proteinase K digestion, which it was achieved using 1 ml per well of a 10 mg/ml crude Proteinase K (Sigma) in Proteinase K buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM CaCl2 , 1 mM MgCl2 , 1 mM KCl) for 5 hours at 16 °C, after which cells were detached from the growth surface.

    Techniques: Injection, Expressing, Infection, Fluorescence

    Effect of Proteinase K on biofilms and measurement of extracellular protease activity in AIP-detached biofilms. (A) Proteinase K (proK, 2 µg/ml) was added to a 2 day old biofilm (strain AH500) and the biofilm integrity was monitored with CLSM at 6 and 12 hr. (B) Levels of protease activity detected in biofilm effluent collected from wild type (SH1000) or Δ agr (SH1001) biofilms supplemented with indicated AIP's. Protease activity was referenced to wild-type without AIP addition. (C) The effect of inhibitors or activators on the proteolytic activity in an AIP-I detached biofilm effluent. Activity assay was supplemented with either the metalloprotease inhibitor EGTA (1 mM), serine protease inhibitor PMSF (10 µM), or the reducing agent DTT (1 mM). Error bars show SEM.

    Journal: PLoS Pathogens

    Article Title: agr-Mediated Dispersal of Staphylococcus aureus Biofilms

    doi: 10.1371/journal.ppat.1000052

    Figure Lengend Snippet: Effect of Proteinase K on biofilms and measurement of extracellular protease activity in AIP-detached biofilms. (A) Proteinase K (proK, 2 µg/ml) was added to a 2 day old biofilm (strain AH500) and the biofilm integrity was monitored with CLSM at 6 and 12 hr. (B) Levels of protease activity detected in biofilm effluent collected from wild type (SH1000) or Δ agr (SH1001) biofilms supplemented with indicated AIP's. Protease activity was referenced to wild-type without AIP addition. (C) The effect of inhibitors or activators on the proteolytic activity in an AIP-I detached biofilm effluent. Activity assay was supplemented with either the metalloprotease inhibitor EGTA (1 mM), serine protease inhibitor PMSF (10 µM), or the reducing agent DTT (1 mM). Error bars show SEM.

    Article Snippet: For the Proteinase K detachment experiments, the enzyme (Sigma-Aldrich) was suspended in water and added to the media reservoir at a final concentration of 2 µg/ml.

    Techniques: Activity Assay, Confocal Laser Scanning Microscopy, Protease Inhibitor

    Effect of treatment of ECP from P. atlantica with proteinase K (PK) (ratio, 5:1) for 60 min on the number of circulating hemocytes per milliliter of blood (THC). *, P

    Journal: Applied and Environmental Microbiology

    Article Title: Effect of Extracellular Products of Pseudoalteromonas atlantica on the Edible Crab Cancer pagurus

    doi: 10.1128/AEM.70.2.729-735.2004

    Figure Lengend Snippet: Effect of treatment of ECP from P. atlantica with proteinase K (PK) (ratio, 5:1) for 60 min on the number of circulating hemocytes per milliliter of blood (THC). *, P

    Article Snippet: For proteinase K treatment of P. atlantica ECP, ECP was mixed at an ECP/proteinase K ratio of either 6:1 or 5:1 with a solution containing 2.5 mg of proteinase K (Sigma Chemical Co., Poole, United Kingdom) per ml and incubated at 60°C for 1 h. As a control, ECP was replaced in the reaction mixture by distilled water.

    Techniques:

    Coomassie blue-stained SDS-PAGE gels of molecular weight markers (lane 1), undiluted ECP (lane 2), ECP treated with proteinase K (ratio, 5:1) for 60 min (lane 3), and ECP treated with proteinase K (ratio, 6:1) for 60 min (lane 4).

    Journal: Applied and Environmental Microbiology

    Article Title: Effect of Extracellular Products of Pseudoalteromonas atlantica on the Edible Crab Cancer pagurus

    doi: 10.1128/AEM.70.2.729-735.2004

    Figure Lengend Snippet: Coomassie blue-stained SDS-PAGE gels of molecular weight markers (lane 1), undiluted ECP (lane 2), ECP treated with proteinase K (ratio, 5:1) for 60 min (lane 3), and ECP treated with proteinase K (ratio, 6:1) for 60 min (lane 4).

    Article Snippet: For proteinase K treatment of P. atlantica ECP, ECP was mixed at an ECP/proteinase K ratio of either 6:1 or 5:1 with a solution containing 2.5 mg of proteinase K (Sigma Chemical Co., Poole, United Kingdom) per ml and incubated at 60°C for 1 h. As a control, ECP was replaced in the reaction mixture by distilled water.

    Techniques: Staining, SDS Page, Molecular Weight

    Enzymatic activity of chitinase is required for regulation of Fn biofilm formation. ( A ) Effect of exogenously added chitinase on biofilm formation in the negatively-charged TC plates. EC 50 s of exogenous chitinase to WT, chiA and chiB mutants were determined to be 0.65, 0.18, and 0.21 μg/ml, respectively (n = 6). ( B ) Effect of exogenous chitinase on biofilm formation in the positively-charged amine plates. EC 50 s of chitinase to WT, chiA and chiB mutants were determined to be 87.46, 0.17, and 0.15 μg/ml, respectively (n = 6). ( C ) Detachment of Fn biofilms after exposure to proteinase K, chitinase and DNase I (50 μg/ml) in the TC plates. Untreated control CV 570 values were 0.149±0.032, 0.588±0.012, and 0.585±0.017 for Fn WT, chiA and chiB mutants, respectively. *P

    Journal: PLoS ONE

    Article Title: Chitinases Are Negative Regulators of Francisella novicida Biofilms

    doi: 10.1371/journal.pone.0093119

    Figure Lengend Snippet: Enzymatic activity of chitinase is required for regulation of Fn biofilm formation. ( A ) Effect of exogenously added chitinase on biofilm formation in the negatively-charged TC plates. EC 50 s of exogenous chitinase to WT, chiA and chiB mutants were determined to be 0.65, 0.18, and 0.21 μg/ml, respectively (n = 6). ( B ) Effect of exogenous chitinase on biofilm formation in the positively-charged amine plates. EC 50 s of chitinase to WT, chiA and chiB mutants were determined to be 87.46, 0.17, and 0.15 μg/ml, respectively (n = 6). ( C ) Detachment of Fn biofilms after exposure to proteinase K, chitinase and DNase I (50 μg/ml) in the TC plates. Untreated control CV 570 values were 0.149±0.032, 0.588±0.012, and 0.585±0.017 for Fn WT, chiA and chiB mutants, respectively. *P

    Article Snippet: Enzymatic detachment of biofilms Proteinase K from Trichoderma album, chitinase from Streptomyces griseus, and DNase I (Sigma-Aldrich) were used.

    Techniques: Activity Assay

    Changes in fibril water accessibility and fibril degradation. ( a – c ) Water-edited solid-state NMR  13 C- 13 C correlation spectra of ( a ) αS and ( b ) co-incubated αS/βS fibrils. Magnetization was equilibrated at long water spin-diffusion times (100 ms,  black ) compared with the initial water-protein magnetization transfer at short spin-diffusion times (3 ms,  red ). ( c ) 1D slices taken at the blue dashed lines in ( a ) and ( b ) of αS fibrils (left side) and αS/βS fibrils (right side), showing the intensities of cross-peaks to lysine (top) or valine (bottom) side chains. The ratio of the cross-peak intensities at long and short spin diffusion times (Int 3ms /Int 100ms ) indicates the relative proximity of water on a residue-specific basis. ( d ) Digestion of αS and αS/βS fibrils at various concentrations of proteinase K. Full-length gels are presented in Supplementary Fig.   6 . ( e ) Map of the residues that show the largest degree of change in water accessibility between αS and αS/βS fibrils, lysine (blue) and threonine (green), on the core-residues (44–96) of PDB structure 2N0A.

    Journal: Scientific Reports

    Article Title: Increased Dynamics of α-Synuclein Fibrils by β-Synuclein Leads to Reduced Seeding and Cytotoxicity

    doi: 10.1038/s41598-019-54063-8

    Figure Lengend Snippet: Changes in fibril water accessibility and fibril degradation. ( a – c ) Water-edited solid-state NMR 13 C- 13 C correlation spectra of ( a ) αS and ( b ) co-incubated αS/βS fibrils. Magnetization was equilibrated at long water spin-diffusion times (100 ms, black ) compared with the initial water-protein magnetization transfer at short spin-diffusion times (3 ms, red ). ( c ) 1D slices taken at the blue dashed lines in ( a ) and ( b ) of αS fibrils (left side) and αS/βS fibrils (right side), showing the intensities of cross-peaks to lysine (top) or valine (bottom) side chains. The ratio of the cross-peak intensities at long and short spin diffusion times (Int 3ms /Int 100ms ) indicates the relative proximity of water on a residue-specific basis. ( d ) Digestion of αS and αS/βS fibrils at various concentrations of proteinase K. Full-length gels are presented in Supplementary Fig.  6 . ( e ) Map of the residues that show the largest degree of change in water accessibility between αS and αS/βS fibrils, lysine (blue) and threonine (green), on the core-residues (44–96) of PDB structure 2N0A.

    Article Snippet: Proteinase K digestion Fibrils at a concentration of 1 mg/mL were incubated with various concentrations (0.1, 0.5, 1.0, 2.0, 5.0 μg/mL) of proteinase K (Sigma Aldrich, St. Louis, MO) in 10 mM PBS (pH 7.4) at 37 °C for 1 h. The digestion reaction was quenched by the addition of a 1200:1 molar excess of phenylmethane sulfonyl fluoride (Sigma Aldrich, St. Louis, MO) followed by the addition of 2 M guanidine thiocyanate (Sigma Aldrich, St. Louis, MO) and incubation at room temperature for 4 h. The results of the degradation reaction were mixed with 4x SDS-PAGE loading buffer (Invitrogen, Carlsbad, CA), loaded onto precast gels (Bio-Rad, Hercules, CA), and run at 120 V for 50 min.

    Techniques: Nuclear Magnetic Resonance, Incubation, Diffusion-based Assay, Mass Spectrometry

    Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested  with  Sac I (lanes 3 and 4), and  Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3,  Sac I-treated pELF1 excised from PFGE gel; 4,  Sac I-treated pELF1 excised from SDS-PFGE gel; 5,  Sma I-treated pELF1 excised from PFGE gel; 6,  Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.

    Journal: Frontiers in Microbiology

    Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

    doi: 10.3389/fmicb.2019.02568

    Figure Lengend Snippet: Retardation assay of pELF1 DNAs excised from PFGE or SDS-PFGE gels. DNA bands of pELF1 were excised from PFGE gel (proteinase K treatment +) or SDS-PFGE gel (proteinase K treatment –), and digested with Sac I (lanes 3 and 4), and Sma I (lanes 5 and 6). After digestion, the samples were subjected to PFGE. MM, Low Range PFG Marker; 1, pELF1 excised from PFGE gel; 2, pELF1 excised from SDS-PFGE gel; 3, Sac I-treated pELF1 excised from PFGE gel; 4, Sac I-treated pELF1 excised from SDS-PFGE gel; 5, Sma I-treated pELF1 excised from PFGE gel; 6, Sma I-treated pELF1 excised from SDS-PFGE gel. N. D.; not digested.

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

    PFGE of the restriction fragments of pELF1 with proteinase K treatment. Lanes: Low Range PFG Marker; proteinase K-treatment only; proteinase K-treated Sal I digests; proteinase K-treated Sma I digests; proteinase K-treated Eag I digests. N. D., not digested.

    Journal: Frontiers in Microbiology

    Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

    doi: 10.3389/fmicb.2019.02568

    Figure Lengend Snippet: PFGE of the restriction fragments of pELF1 with proteinase K treatment. Lanes: Low Range PFG Marker; proteinase K-treatment only; proteinase K-treated Sal I digests; proteinase K-treated Sma I digests; proteinase K-treated Eag I digests. N. D., not digested.

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

    PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 without proteinase K treatment; AA708 with proteinase K treatment; AA708 with both proteinase K and S1 nuclease treatment.

    Journal: Frontiers in Microbiology

    Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

    doi: 10.3389/fmicb.2019.02568

    Figure Lengend Snippet: PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 without proteinase K treatment; AA708 with proteinase K treatment; AA708 with both proteinase K and S1 nuclease treatment.

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

    SDS-PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 with proteinase K treatment; AA708 without proteinase K treatment; AA708 with proteinase K and S1 nuclease treatment.

    Journal: Frontiers in Microbiology

    Article Title: Novel Multidrug-Resistant Enterococcal Mobile Linear Plasmid pELF1 Encoding vanA and vanM Gene Clusters From a Japanese Vancomycin-Resistant Enterococci Isolate

    doi: 10.3389/fmicb.2019.02568

    Figure Lengend Snippet: SDS-PFGE of the AA708 strain. Lanes: Low Range PFG Marker; AA708 with proteinase K treatment; AA708 without proteinase K treatment; AA708 with proteinase K and S1 nuclease treatment.

    Article Snippet: Agarose plugs (1%) containing embedded enterococci were treated with lysozyme (Roche Diagnostics K.K, Minneapolis, MN, United States) solution (10 mg/ml) at 37°C for 6 h, followed by treatment with proteinase K (Merck Millipore, Darmstadt, Germany) solution (60 mAnson U/ml) at 50°C for 48 h. After washing the plugs with wash buffer (20 mM Tris-HCl, pH 8.0; 50 mM EDTA), proteinase K was inhibited using phenylmethylsulfonyl fluoride (PMSF) solution (20 mM Tris-HCl, pH 8.0, 50 mM EDTA, and 1 mM PMSF).

    Techniques: Marker

    In vivo  activation of DCs after U-Omp19 injection. U-Omp19 either untreated or digested with proteinase K,  E. coli  LPS or PBS alone was injected i.v. to BALB/c mice. Splenic CD11c +  DCs were analyzed for their activation status 20 h after injection by assessing the surface expression of ( A ) CD40, ( B ) CD80 and ( C ) CD86 molecules by flow cytometry. This experiment was conducted three times with similar results. Histograms display results from one representative experiment.

    Journal: PLoS ONE

    Article Title: An Oral Vaccine Based on U-Omp19 Induces Protection against B. abortus Mucosal Challenge by Inducing an Adaptive IL-17 Immune Response in Mice

    doi: 10.1371/journal.pone.0016203

    Figure Lengend Snippet: In vivo activation of DCs after U-Omp19 injection. U-Omp19 either untreated or digested with proteinase K, E. coli LPS or PBS alone was injected i.v. to BALB/c mice. Splenic CD11c + DCs were analyzed for their activation status 20 h after injection by assessing the surface expression of ( A ) CD40, ( B ) CD80 and ( C ) CD86 molecules by flow cytometry. This experiment was conducted three times with similar results. Histograms display results from one representative experiment.

    Article Snippet: U-Omp19 was treated with proteinase K-agarose from Tricirachium album (Sigma) for 2 h at 37°C following manufacturer's indications.

    Techniques: In Vivo, Activation Assay, Injection, Mouse Assay, Expressing, Flow Cytometry, Cytometry

    A protenious antibacterial activity is present in the serum 24 h after immunization with the EV76 live vaccine strain. (A) Sera collected from EV76-immunized mice at the indicated time points post-infection (1 h–13 d) were analyzed by the Y. pestis ex vivo growth assays. (B) Serum samples were collected from saline-injected or EV76-immunized mice at the indicated time points p.i. (24 h–4 d), were serial diluted and analyzed for affecting Y. pestis growth by ex vivo assays. (C) Serum derived from control and EV76-immunized mice was subjected to proteinase K digestion and used for Y. pestis ex vivo growth assays. In all of the experiments, the data depict the mean and the standard error of the mean (SEM) of at least 3 individual sera, of at least two independent experiments. Statistical significance was measured using Student's unpaired t -test with log-transformed values ( **** p

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: Host Iron Nutritional Immunity Induced by a Live Yersinia pestis Vaccine Strain Is Associated with Immediate Protection against Plague

    doi: 10.3389/fcimb.2017.00277

    Figure Lengend Snippet: A protenious antibacterial activity is present in the serum 24 h after immunization with the EV76 live vaccine strain. (A) Sera collected from EV76-immunized mice at the indicated time points post-infection (1 h–13 d) were analyzed by the Y. pestis ex vivo growth assays. (B) Serum samples were collected from saline-injected or EV76-immunized mice at the indicated time points p.i. (24 h–4 d), were serial diluted and analyzed for affecting Y. pestis growth by ex vivo assays. (C) Serum derived from control and EV76-immunized mice was subjected to proteinase K digestion and used for Y. pestis ex vivo growth assays. In all of the experiments, the data depict the mean and the standard error of the mean (SEM) of at least 3 individual sera, of at least two independent experiments. Statistical significance was measured using Student's unpaired t -test with log-transformed values ( **** p

    Article Snippet: Proteinase K digestion Proteolysis was performed by incubating the serum samples with proteinase K-conjugated beads (Sigma Aldrich P9290) for 2 h at 37°C according to the manufacturer's recommendations.

    Techniques: Activity Assay, Mouse Assay, Infection, Ex Vivo, Injection, Derivative Assay, Transformation Assay

    Detection of CDK2 in HBV capsids purified from HepG2 cells by Western blotting. HBV capsids (0.5 μg) were digested with proteinase K (PK) agarose beads as described in Materials and Methods. Proteinase K was then inactivated by the addition of the proteinase K inhibitor, and the sample was resolved by SDS-PAGE (lanes 8 and 10), along with the same amount of undigested capsids from the same fractions (lanes 7 and 9). Purified GST-CDK2 standards (lanes 1 to 3) and total lysate from HepG2 cells (lane 4) were loaded as controls for CDK2 detection. A sucrose fraction that did not contain HBV capsids was also treated with the proteinase K agarose beads to ensure that contaminating proteins were removed by the digestion (lane 6) or mock treated (lane 5). The capsids loaded in lanes 9 and 10 were from a capsid-positive sucrose fraction of higher purity than those loaded in lanes 7 and 8. CDK2 was detected by Western blotting using the anti-CDK2 antibody (top panel). HBc was visualized by Sypro ruby staining (bottom panel). C, core protein.

    Journal: Journal of Virology

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

    doi: 10.1128/JVI.01218-12

    Figure Lengend Snippet: Detection of CDK2 in HBV capsids purified from HepG2 cells by Western blotting. HBV capsids (0.5 μg) were digested with proteinase K (PK) agarose beads as described in Materials and Methods. Proteinase K was then inactivated by the addition of the proteinase K inhibitor, and the sample was resolved by SDS-PAGE (lanes 8 and 10), along with the same amount of undigested capsids from the same fractions (lanes 7 and 9). Purified GST-CDK2 standards (lanes 1 to 3) and total lysate from HepG2 cells (lane 4) were loaded as controls for CDK2 detection. A sucrose fraction that did not contain HBV capsids was also treated with the proteinase K agarose beads to ensure that contaminating proteins were removed by the digestion (lane 6) or mock treated (lane 5). The capsids loaded in lanes 9 and 10 were from a capsid-positive sucrose fraction of higher purity than those loaded in lanes 7 and 8. CDK2 was detected by Western blotting using the anti-CDK2 antibody (top panel). HBc was visualized by Sypro ruby staining (bottom panel). C, core protein.

    Article Snippet: HepG2 HBV capsids in sucrose fractions were digested for 1.5 h with proteinase K-agarose beads (Sigma) at 37°C with gentle agitation.

    Techniques: Purification, Western Blot, SDS Page, Staining

    Endogenous kinase reactions with virion-derived HBV capsids. HBV capsids were released from virions purified from the medium of HBV-transfected HepG2 cells by NP-40 treatment. One control aliquot was removed, and the remaining virion-derived capsids were digested by proteinase K. The untreated (lane 1) or proteinase K-treated (lanes 2 to 5), virion-derived capsids (ca. 6 ng per reaction) were phosphorylated by the encapsidated kinase in endogenous kinase reactions in the presence of the indicated kinase inhibitors (lanes 3 to 5) or DMSO control (lane 2). Kinase inhibitors used included CDK2 inhibitor III (lane 3, 250 μM, 500× IC 50 for CDK2), roscovitine (lane 4, 250 μM, 350× IC 50 for CDK2), and Bisindo (lane 3, 5 μM, 500× IC 50 for PKC). An equal amount of proteinase K alone was also loaded as an additional control (lane 6). In parallel, 6 ng (lane 7) or 12 ng (lane 8) of cytoplasmic HBV capsids from HepG2 cells (also treated with proteinase K; see Materials and Methods) were subjected to the endogenous kinase reaction. Half of each reaction product was resolved by agarose gel electrophoresis to visualize capsid particles (top), and proteinase K inhibitor was added to the other half to terminate the digestion before boiling in SDS sample buffer and resolving by SDS-PAGE to visualize the total core protein (bottom). The gels were dried, and labeled capsids or core proteins were detected by autoradiography. *, unknown labeled species. CA, capsids; C, core protein; PK, proteinase K; IC, intracellular.

    Journal: Journal of Virology

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids

    doi: 10.1128/JVI.01218-12

    Figure Lengend Snippet: Endogenous kinase reactions with virion-derived HBV capsids. HBV capsids were released from virions purified from the medium of HBV-transfected HepG2 cells by NP-40 treatment. One control aliquot was removed, and the remaining virion-derived capsids were digested by proteinase K. The untreated (lane 1) or proteinase K-treated (lanes 2 to 5), virion-derived capsids (ca. 6 ng per reaction) were phosphorylated by the encapsidated kinase in endogenous kinase reactions in the presence of the indicated kinase inhibitors (lanes 3 to 5) or DMSO control (lane 2). Kinase inhibitors used included CDK2 inhibitor III (lane 3, 250 μM, 500× IC 50 for CDK2), roscovitine (lane 4, 250 μM, 350× IC 50 for CDK2), and Bisindo (lane 3, 5 μM, 500× IC 50 for PKC). An equal amount of proteinase K alone was also loaded as an additional control (lane 6). In parallel, 6 ng (lane 7) or 12 ng (lane 8) of cytoplasmic HBV capsids from HepG2 cells (also treated with proteinase K; see Materials and Methods) were subjected to the endogenous kinase reaction. Half of each reaction product was resolved by agarose gel electrophoresis to visualize capsid particles (top), and proteinase K inhibitor was added to the other half to terminate the digestion before boiling in SDS sample buffer and resolving by SDS-PAGE to visualize the total core protein (bottom). The gels were dried, and labeled capsids or core proteins were detected by autoradiography. *, unknown labeled species. CA, capsids; C, core protein; PK, proteinase K; IC, intracellular.

    Article Snippet: HepG2 HBV capsids in sucrose fractions were digested for 1.5 h with proteinase K-agarose beads (Sigma) at 37°C with gentle agitation.

    Techniques: Derivative Assay, Purification, Transfection, Agarose Gel Electrophoresis, SDS Page, Labeling, Autoradiography