Structured Review

Promega proteinase k
Integrin engagement and activation of the integrin/Src/paxillin signaling pathway by the gH/gL/UL128-131 complex allows efficient HCMV internalization into target monocytes. (A and B) Monocytes were pretreated with 1 µM PP2, 1 µM AG1478, 5 µg/ml of blocking anti-β1 or anti-β3 integrin antibodies, or 5 µg/ml of IgG for 1 h at 37°C/5% CO 2 . (C and D) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (A, B, C, and D) Monocytes were then HCMV (BADwt or BADrUL131)-infected (M.O.I. of 0.1) for 1 h at 4°C, washed, treated with 5 U/ml of α-thrombin (D only), then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with <t>Proteinase</t> K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p
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1) Product Images from "The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes"

Article Title: The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1003463

Integrin engagement and activation of the integrin/Src/paxillin signaling pathway by the gH/gL/UL128-131 complex allows efficient HCMV internalization into target monocytes. (A and B) Monocytes were pretreated with 1 µM PP2, 1 µM AG1478, 5 µg/ml of blocking anti-β1 or anti-β3 integrin antibodies, or 5 µg/ml of IgG for 1 h at 37°C/5% CO 2 . (C and D) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (A, B, C, and D) Monocytes were then HCMV (BADwt or BADrUL131)-infected (M.O.I. of 0.1) for 1 h at 4°C, washed, treated with 5 U/ml of α-thrombin (D only), then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p
Figure Legend Snippet: Integrin engagement and activation of the integrin/Src/paxillin signaling pathway by the gH/gL/UL128-131 complex allows efficient HCMV internalization into target monocytes. (A and B) Monocytes were pretreated with 1 µM PP2, 1 µM AG1478, 5 µg/ml of blocking anti-β1 or anti-β3 integrin antibodies, or 5 µg/ml of IgG for 1 h at 37°C/5% CO 2 . (C and D) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (A, B, C, and D) Monocytes were then HCMV (BADwt or BADrUL131)-infected (M.O.I. of 0.1) for 1 h at 4°C, washed, treated with 5 U/ml of α-thrombin (D only), then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

Techniques Used: Activation Assay, Blocking Assay, Transfection, Infection, Real-time Polymerase Chain Reaction

Presence of the gH/gL/UL128-131 complex links HCMV′s ability to activate and efficiently enter into target monocytes. (A) Approximately 2×10 6 virions of Towne (p.40, p.51 and p.57), AD169, TB40/E and TB40/F were spun down through a sucrose cushion, lysed and western blot analyses were performed using antibodies recognizing the HCMV proteins, pp65 and pUL130. (B) Monocytes were isolated and cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src and p70 S6 kinase. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (C) Monocytes were mock infected or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169) infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and semi-quantitative PCR was performed using primers complementary to genomic HCMV DNA and cellular GAPDH, as an internal control. PCR reactions were analyzed by agarose gel electrophoresis using ethidium bromide. (D, E, F, G, H, I and J) Monocytes were mock- or HCMV (TB40/E or AD169)-infected (M.O.I. of 5) for 1 h at 4°C, then 2 mM of DTSSP [3,3′-dithiobis (sulfosuccinimidylpropionate] was added at 4°C for additional 2 h. Cells were spun down and lysed. Antibodies recognizing β1, β3 integrins, HCMV gH or isotype control IgG were added overnight at 4°C to cellular lysates and then protein A/G Sepharose was added for 4 h at 4°C. Protein A/G Sepharose beads with bound protein complexes were spun down, washed with a lysis buffer and resuspended in sample buffer. Western blot analyses were performed using antibodies recognizing β1 and β3 integrins, as well as the HCMV gH and pUL130. Lysates from HCMV-infected monocytes were also analyzed for equal levels of β1, β3 integrins and actin in samples undergoing immunoprecipitation. All experiments were repeated at least three times and representative results are shown. Note: The arrows point to the band of interest. The asterisks mark non-specific bands. (K) The schematic diagram describes our cumulative data from the immunoprecipitation analysis; illustrating the interaction between the gH/gL/UL128-131 complex of TB40/E strain or gH/gL/(gO) complex of AD169 strain with cellular integrins.
Figure Legend Snippet: Presence of the gH/gL/UL128-131 complex links HCMV′s ability to activate and efficiently enter into target monocytes. (A) Approximately 2×10 6 virions of Towne (p.40, p.51 and p.57), AD169, TB40/E and TB40/F were spun down through a sucrose cushion, lysed and western blot analyses were performed using antibodies recognizing the HCMV proteins, pp65 and pUL130. (B) Monocytes were isolated and cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src and p70 S6 kinase. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (C) Monocytes were mock infected or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169) infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and semi-quantitative PCR was performed using primers complementary to genomic HCMV DNA and cellular GAPDH, as an internal control. PCR reactions were analyzed by agarose gel electrophoresis using ethidium bromide. (D, E, F, G, H, I and J) Monocytes were mock- or HCMV (TB40/E or AD169)-infected (M.O.I. of 5) for 1 h at 4°C, then 2 mM of DTSSP [3,3′-dithiobis (sulfosuccinimidylpropionate] was added at 4°C for additional 2 h. Cells were spun down and lysed. Antibodies recognizing β1, β3 integrins, HCMV gH or isotype control IgG were added overnight at 4°C to cellular lysates and then protein A/G Sepharose was added for 4 h at 4°C. Protein A/G Sepharose beads with bound protein complexes were spun down, washed with a lysis buffer and resuspended in sample buffer. Western blot analyses were performed using antibodies recognizing β1 and β3 integrins, as well as the HCMV gH and pUL130. Lysates from HCMV-infected monocytes were also analyzed for equal levels of β1, β3 integrins and actin in samples undergoing immunoprecipitation. All experiments were repeated at least three times and representative results are shown. Note: The arrows point to the band of interest. The asterisks mark non-specific bands. (K) The schematic diagram describes our cumulative data from the immunoprecipitation analysis; illustrating the interaction between the gH/gL/UL128-131 complex of TB40/E strain or gH/gL/(gO) complex of AD169 strain with cellular integrins.

Techniques Used: Western Blot, Isolation, Cell Culture, Infection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Lysis, Immunoprecipitation

Presence of the gH/gL/UL128-131 complex has no effect on the integrin/Src/paxillin signaling pathway in and HCMV internalization into fibroblasts. (A) Fibroblasts were cultured in low serum for 24 h at 37°C/5% CO 2 . Fibroblasts were then mock- or HCMV (BADwt or BADrUL131)-infected (M.O.I. of 5) and harvested at 20 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, and Erk. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then left at 4°C or temperature shifted to 37°C for 1 h. (C) Fibroblasts were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. (B and C) Then, fibroblasts were washed and treated with Proteinase K solution for 1 h. Cells were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p
Figure Legend Snippet: Presence of the gH/gL/UL128-131 complex has no effect on the integrin/Src/paxillin signaling pathway in and HCMV internalization into fibroblasts. (A) Fibroblasts were cultured in low serum for 24 h at 37°C/5% CO 2 . Fibroblasts were then mock- or HCMV (BADwt or BADrUL131)-infected (M.O.I. of 5) and harvested at 20 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, and Erk. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then left at 4°C or temperature shifted to 37°C for 1 h. (C) Fibroblasts were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. (B and C) Then, fibroblasts were washed and treated with Proteinase K solution for 1 h. Cells were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

Techniques Used: Cell Culture, Infection, Western Blot, Transfection, Real-time Polymerase Chain Reaction

Regulation of the actin cytoskeleton and dynamin is essential for efficient internalization of clinical-like HCMV isolates into monocytes, but not into fibroblasts. (A) Monocytes were pretreated with DMSO, 0.5 µM jasplakinolide, or 2.5 µM latrunculin A. (B) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (C) Monocytes were preatreated with 50 µM dynasore. (D) Monocytes were transfected with siRNA complementary to dynamin or a control siRNA for 48 h. (E and F) Fibroblasts were pretreated with DMSO, 0.5 µM jasplakinolide, 2.5 µM latrunculin A, or 50 µM dynasore. (A, B, C, D, E, and F) Cells were then infected with BADwt, BADrUL131 or TB40/E (M.O.I. of 0.1) for 1 h at 4°C washed and then temperature shifted to 37°C for 1 h. Monocytes were then treated with 0.5 µM jasplakinolide at 37°C for an additional 1 h (only B). Cells were then washed and treated with Proteinase K solution for 1 h. Cells were harvested and PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA. For qPCR data, results are plotted as a mean ±SEM. Student's T-tests were performed and p
Figure Legend Snippet: Regulation of the actin cytoskeleton and dynamin is essential for efficient internalization of clinical-like HCMV isolates into monocytes, but not into fibroblasts. (A) Monocytes were pretreated with DMSO, 0.5 µM jasplakinolide, or 2.5 µM latrunculin A. (B) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (C) Monocytes were preatreated with 50 µM dynasore. (D) Monocytes were transfected with siRNA complementary to dynamin or a control siRNA for 48 h. (E and F) Fibroblasts were pretreated with DMSO, 0.5 µM jasplakinolide, 2.5 µM latrunculin A, or 50 µM dynasore. (A, B, C, D, E, and F) Cells were then infected with BADwt, BADrUL131 or TB40/E (M.O.I. of 0.1) for 1 h at 4°C washed and then temperature shifted to 37°C for 1 h. Monocytes were then treated with 0.5 µM jasplakinolide at 37°C for an additional 1 h (only B). Cells were then washed and treated with Proteinase K solution for 1 h. Cells were harvested and PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA. For qPCR data, results are plotted as a mean ±SEM. Student's T-tests were performed and p

Techniques Used: Transfection, Infection, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

The HCMV gH/gL/UL128-131 complex is critical for activation of, and for efficient and productive viral internalization into target monocytes. (A) Monocytes were cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (BAD wt or BAD r UL131)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, Erk and SAPK/JNK. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Monocytes were HCMV (BADwt, BADrUL131, TB40/F or TB40/E)-infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p
Figure Legend Snippet: The HCMV gH/gL/UL128-131 complex is critical for activation of, and for efficient and productive viral internalization into target monocytes. (A) Monocytes were cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (BAD wt or BAD r UL131)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, Erk and SAPK/JNK. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Monocytes were HCMV (BADwt, BADrUL131, TB40/F or TB40/E)-infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

Techniques Used: Activation Assay, Cell Culture, Infection, Western Blot, Real-time Polymerase Chain Reaction

2) Product Images from "Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1"

Article Title: Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt588

Inhibition of PolB1-mediated DNA strand displacement by Sso7d. ( A ) Effect of Sso7d on strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Sso7d. The reaction mixtures were treated with proteinase K and extracted with phenol/chloroform. Samples were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–9, Sso7d was added to 0, 0.5, 2.5, 10, 25, 50 and 90 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on templates bound maximally by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 or P36/L72 (2 nM) in the presence of 90 μM Sso7d. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. ( C ) Sketches of primer templates used in experiments shown in Figure 2 D. Primers P17 and P59 are annealed to C72 starting from the same base on the minicircle as that for P36. P36(5′AT-rich) and P36(5′GC-rich), which carry 5′-AT-rich and 5′-GC-rich sequences, respectively, are annealed to different regions of C72. ( D ) Template dependence of the inhibition of strand displacement by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated primer template (2 nM) in the presence or absence of Sso7d (90 μM). Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Gels were dried and exposed to radiographic film.
Figure Legend Snippet: Inhibition of PolB1-mediated DNA strand displacement by Sso7d. ( A ) Effect of Sso7d on strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Sso7d. The reaction mixtures were treated with proteinase K and extracted with phenol/chloroform. Samples were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–9, Sso7d was added to 0, 0.5, 2.5, 10, 25, 50 and 90 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on templates bound maximally by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 or P36/L72 (2 nM) in the presence of 90 μM Sso7d. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. ( C ) Sketches of primer templates used in experiments shown in Figure 2 D. Primers P17 and P59 are annealed to C72 starting from the same base on the minicircle as that for P36. P36(5′AT-rich) and P36(5′GC-rich), which carry 5′-AT-rich and 5′-GC-rich sequences, respectively, are annealed to different regions of C72. ( D ) Template dependence of the inhibition of strand displacement by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated primer template (2 nM) in the presence or absence of Sso7d (90 μM). Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Gels were dried and exposed to radiographic film.

Techniques Used: Inhibition, Incubation, Electrophoresis, Sequencing

Modulation of PolB1-mediated strand displacement by Sso7d in the presence of PCNA and RFC. ( A ) PolB1-mediated DNA strand displacement. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Sso7d were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–10, Sso7d concentrations were 0.5, 2.5, 10, 25, 50, 90 μM, respectively. ( B ) PolB1-mediated RNA strand displacement. Reactions were assembled and processed as described in (A) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.
Figure Legend Snippet: Modulation of PolB1-mediated strand displacement by Sso7d in the presence of PCNA and RFC. ( A ) PolB1-mediated DNA strand displacement. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Sso7d were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–10, Sso7d concentrations were 0.5, 2.5, 10, 25, 50, 90 μM, respectively. ( B ) PolB1-mediated RNA strand displacement. Reactions were assembled and processed as described in (A) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

Techniques Used: Electrophoresis

Effect of Sso7d on RNA strand displacement by PolB1. ( A ) Sketches of RNA-primed templates used in experiments shown in Figure 4 B and C. The 12-nt oligoribonucleotide portion of the primer was indicated with a wavy line. ( B ) RNA strand displacement by PolB1 on P36(5′RNA)/C72 in the presence of Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Sso7d. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–8, Sso7d was added to 0, 0.5, 2.5, 10, 25 and 90 μM, respectively. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. ( C ) Sizes of the products of strand displacement by PolB1 in the presence of 90 μM Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated template (2 nM) in the presence of Sso7d (90 μM) under the standard assay conditions. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. Gels were dried and exposed to radiographic film.
Figure Legend Snippet: Effect of Sso7d on RNA strand displacement by PolB1. ( A ) Sketches of RNA-primed templates used in experiments shown in Figure 4 B and C. The 12-nt oligoribonucleotide portion of the primer was indicated with a wavy line. ( B ) RNA strand displacement by PolB1 on P36(5′RNA)/C72 in the presence of Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Sso7d. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–8, Sso7d was added to 0, 0.5, 2.5, 10, 25 and 90 μM, respectively. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. ( C ) Sizes of the products of strand displacement by PolB1 in the presence of 90 μM Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated template (2 nM) in the presence of Sso7d (90 μM) under the standard assay conditions. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. Gels were dried and exposed to radiographic film.

Techniques Used: Incubation, Electrophoresis, Sequencing

Comparison between Cren7 and Sso7d in modulating strand displacement by PolB1. ( A ) Effect of Cren7 on DNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Cren7. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 0.64 μM Cren7; lanes 3–9, Cren7 was added to 0, 0.04, 0.08, 0.12, 0.16, 0.32 and 0.64 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on P36/L72 and P36/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated with P36/L72 or P36/C72 (2 nM) in the presence of 0.64 μM Cren7 under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1× TBE. ( C ) Effect of Cren7 on RNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Cren7. Lane 1, control; lane 2, 2.5 μM Cren7; lanes 3–10, Cren7 was added to 0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1 × TBE. ( D ) Sizes of the products of strand displacement by PolB1 on P36(5′RNA)/L72 and P36(5′RNA)/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/L72 or P36(5′RNA)/C72 (2 nM) in the presence of Cren7 (2.5 μM) under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1 × TBE. ( E ) Modulation of PolB1-mediated DNA strand displacement by Cren7 in the presence of PCNA and RFC. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Cren7 were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–11, Cren7 concentrations were 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. ( F ) Modulation of PolB1-mediated RNA strand displacement by Cren7 in the presence of PCNA and RFC. Reactions were assembled and processed as described in (E) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.
Figure Legend Snippet: Comparison between Cren7 and Sso7d in modulating strand displacement by PolB1. ( A ) Effect of Cren7 on DNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Cren7. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 0.64 μM Cren7; lanes 3–9, Cren7 was added to 0, 0.04, 0.08, 0.12, 0.16, 0.32 and 0.64 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on P36/L72 and P36/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated with P36/L72 or P36/C72 (2 nM) in the presence of 0.64 μM Cren7 under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1× TBE. ( C ) Effect of Cren7 on RNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Cren7. Lane 1, control; lane 2, 2.5 μM Cren7; lanes 3–10, Cren7 was added to 0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1 × TBE. ( D ) Sizes of the products of strand displacement by PolB1 on P36(5′RNA)/L72 and P36(5′RNA)/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/L72 or P36(5′RNA)/C72 (2 nM) in the presence of Cren7 (2.5 μM) under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1 × TBE. ( E ) Modulation of PolB1-mediated DNA strand displacement by Cren7 in the presence of PCNA and RFC. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Cren7 were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–11, Cren7 concentrations were 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. ( F ) Modulation of PolB1-mediated RNA strand displacement by Cren7 in the presence of PCNA and RFC. Reactions were assembled and processed as described in (E) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

Techniques Used: Incubation, Electrophoresis, Sequencing

3) Product Images from "Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids"

Article Title: Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

Journal: Scientific Reports

doi: 10.1038/srep34048

IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf 21 microsomal membranes. ( a ) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. ( b ) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. ( c–e ) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c ) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. ( d,e ) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14 C-leucine supplementation.
Figure Legend Snippet: IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf 21 microsomal membranes. ( a ) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. ( b ) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. ( c–e ) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c ) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. ( d,e ) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14 C-leucine supplementation.

Techniques Used: Fluorescence, Standard Deviation, Autoradiography, Isotopic Labeling

4) Product Images from "Recombinant Mtb9.8 of Mycobacterium bovis stimulates TNF-α and IL-1β secretion by RAW264.7 macrophages through activation of NF-κB pathway via TLR2"

Article Title: Recombinant Mtb9.8 of Mycobacterium bovis stimulates TNF-α and IL-1β secretion by RAW264.7 macrophages through activation of NF-κB pathway via TLR2

Journal: Scientific Reports

doi: 10.1038/s41598-018-20433-x

rMtb9.8 induces the secretion of TNF-α by THP-1 cells. THP-1 cells were cultured in the presence of rMtb9.8 (5 µg/ml) for 4 h at 37 °C. The cell culture supernatant was collected, and the level of TNF-α in the supernatant was measured using ELISA. Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated rMtb9.8-induced expression of TNF-α (p
Figure Legend Snippet: rMtb9.8 induces the secretion of TNF-α by THP-1 cells. THP-1 cells were cultured in the presence of rMtb9.8 (5 µg/ml) for 4 h at 37 °C. The cell culture supernatant was collected, and the level of TNF-α in the supernatant was measured using ELISA. Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated rMtb9.8-induced expression of TNF-α (p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Positive Control, Plasmid Preparation, Positron Emission Tomography, Negative Control, Expressing

rMtb9.8 induces the secretion of TNF-α ( A ) and IL-1β ( B ) by mouse macrophages. RAW264.7 cells were cultured in the presence of various concentrations of rMtb9.8 (0, 0.5, 1.5, or 5 µg/ml). Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated the rMtb9.8-induced expression of TNF-α and IL-1β (p
Figure Legend Snippet: rMtb9.8 induces the secretion of TNF-α ( A ) and IL-1β ( B ) by mouse macrophages. RAW264.7 cells were cultured in the presence of various concentrations of rMtb9.8 (0, 0.5, 1.5, or 5 µg/ml). Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated the rMtb9.8-induced expression of TNF-α and IL-1β (p

Techniques Used: Cell Culture, Positive Control, Plasmid Preparation, Positron Emission Tomography, Negative Control, Expressing

5) Product Images from "Recombinant Mtb9.8 of Mycobacterium bovis stimulates TNF-α and IL-1β secretion by RAW264.7 macrophages through activation of NF-κB pathway via TLR2"

Article Title: Recombinant Mtb9.8 of Mycobacterium bovis stimulates TNF-α and IL-1β secretion by RAW264.7 macrophages through activation of NF-κB pathway via TLR2

Journal: Scientific Reports

doi: 10.1038/s41598-018-20433-x

rMtb9.8 induces the secretion of TNF-α by THP-1 cells. THP-1 cells were cultured in the presence of rMtb9.8 (5 µg/ml) for 4 h at 37 °C. The cell culture supernatant was collected, and the level of TNF-α in the supernatant was measured using ELISA. Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated rMtb9.8-induced expression of TNF-α (p
Figure Legend Snippet: rMtb9.8 induces the secretion of TNF-α by THP-1 cells. THP-1 cells were cultured in the presence of rMtb9.8 (5 µg/ml) for 4 h at 37 °C. The cell culture supernatant was collected, and the level of TNF-α in the supernatant was measured using ELISA. Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated rMtb9.8-induced expression of TNF-α (p

Techniques Used: Cell Culture, Enzyme-linked Immunosorbent Assay, Positive Control, Plasmid Preparation, Positron Emission Tomography, Negative Control, Expressing

rMtb9.8 induces the secretion of TNF-α ( A ) and IL-1β ( B ) by mouse macrophages. RAW264.7 cells were cultured in the presence of various concentrations of rMtb9.8 (0, 0.5, 1.5, or 5 µg/ml). Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated the rMtb9.8-induced expression of TNF-α and IL-1β (p
Figure Legend Snippet: rMtb9.8 induces the secretion of TNF-α ( A ) and IL-1β ( B ) by mouse macrophages. RAW264.7 cells were cultured in the presence of various concentrations of rMtb9.8 (0, 0.5, 1.5, or 5 µg/ml). Pam3CSK4 (10 µg/ml), a TLR1/2 agonist, was used as a positive control. The pET30 vector tag protein without the Mtb9.8 gene (PET) was used as a negative control. Proteinase K abrogated the rMtb9.8-induced expression of TNF-α and IL-1β (p

Techniques Used: Cell Culture, Positive Control, Plasmid Preparation, Positron Emission Tomography, Negative Control, Expressing

6) Product Images from "Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family"

Article Title: Conserved Features in the Structure, Mechanism, and Biogenesis of the Inverse Autotransporter Protein Family

Journal: Genome Biology and Evolution

doi: 10.1093/gbe/evw112

Structure and assembly of the intimin EaeA. (A) The domain architecture of intimin (i.e., EaeA) is represented based on the PDB structures and homology-modeled domains. The predicted overall structure of intact intimin is colored based on sequence conservation between intimin subtypes (blue = 95% sequence identity; red = 60% sequence identity). (B) Multiple sequence alignments of Tir (showing the region that binds intimin) and of the C-lectin domain of intimin α1, β1, γ1, ϵ1, ζ, θ, ι1, and κ (showing the region that binds Tir). Residues are shaded according to the chemical properties, and contact residues identified in the Tir-intimin crystal structure (pdb 1F02) (sequence indicated with *) are underlined ( supplementary table S3 , Supplementary Material online, has the accession numbers for these intimin and Tir sequences). (C) The Tir:Int complex structure (pdb 1F02) is shown with Tir represented as a stick frame, while the space-filling model of the intimin C-lectin domain is shown in yellow and orange, where orange shading designates residues mediating contact with Tir. (D) Escherichia coli harboring plasmid pCJS30(intimin) were starved of methionine and cysteine, pulse labeled with 35 S-Met and -Cys for 40 s, followed by a chase period with excess 32 S-Met and-Cys, where aliquots were taken at 10 s, 2, 4, 8, 16, and 32 min. Aliquots were incubated with proteinase K (+PK) or without (−PK) for 10 min on ice, followed by TCA-precipitation, acetone washing, and resuspension in SDS loading dye, before analysis by SDS-PAGE. Middle and upper panels, cells also carry the control plasmid pACYCDuet-1. Lower panel, cells carry the complementing plasmid pCJS69( tamA ) (plasmids detailed in supplementary table S4 , Supplementary Material online). The labeled protein band corresponding to the ∼60 kDa TamA is indicated as *.
Figure Legend Snippet: Structure and assembly of the intimin EaeA. (A) The domain architecture of intimin (i.e., EaeA) is represented based on the PDB structures and homology-modeled domains. The predicted overall structure of intact intimin is colored based on sequence conservation between intimin subtypes (blue = 95% sequence identity; red = 60% sequence identity). (B) Multiple sequence alignments of Tir (showing the region that binds intimin) and of the C-lectin domain of intimin α1, β1, γ1, ϵ1, ζ, θ, ι1, and κ (showing the region that binds Tir). Residues are shaded according to the chemical properties, and contact residues identified in the Tir-intimin crystal structure (pdb 1F02) (sequence indicated with *) are underlined ( supplementary table S3 , Supplementary Material online, has the accession numbers for these intimin and Tir sequences). (C) The Tir:Int complex structure (pdb 1F02) is shown with Tir represented as a stick frame, while the space-filling model of the intimin C-lectin domain is shown in yellow and orange, where orange shading designates residues mediating contact with Tir. (D) Escherichia coli harboring plasmid pCJS30(intimin) were starved of methionine and cysteine, pulse labeled with 35 S-Met and -Cys for 40 s, followed by a chase period with excess 32 S-Met and-Cys, where aliquots were taken at 10 s, 2, 4, 8, 16, and 32 min. Aliquots were incubated with proteinase K (+PK) or without (−PK) for 10 min on ice, followed by TCA-precipitation, acetone washing, and resuspension in SDS loading dye, before analysis by SDS-PAGE. Middle and upper panels, cells also carry the control plasmid pACYCDuet-1. Lower panel, cells carry the complementing plasmid pCJS69( tamA ) (plasmids detailed in supplementary table S4 , Supplementary Material online). The labeled protein band corresponding to the ∼60 kDa TamA is indicated as *.

Techniques Used: Sequencing, Plasmid Preparation, Labeling, Incubation, TCA Precipitation, SDS Page

The assembly of FdeC is enhanced by the TAM. (A) The domain architecture of FdeC is represented based on PDB structures and homology-modeled domains. The predicted overall structure of intact FdeC is colored based on the sequence conservation between FdeC subtypes (blue = 95% sequence identity; red = 60% sequence identity). The black lines “N” and “C” encompass the peptides recovered from the 50 kDa fragment and the 100 kDa fragment derived by proteinase K as described in the main text. (B) Escherichia coli harboring plasmid pCJS50( fdeC ) were pulse labeled as in D . Mass spectrometry defined the identity of the 50 kDa N-terminal fragment of FdeC (N), and the 100 kDa C-terminal fragment of FdeC (C).
Figure Legend Snippet: The assembly of FdeC is enhanced by the TAM. (A) The domain architecture of FdeC is represented based on PDB structures and homology-modeled domains. The predicted overall structure of intact FdeC is colored based on the sequence conservation between FdeC subtypes (blue = 95% sequence identity; red = 60% sequence identity). The black lines “N” and “C” encompass the peptides recovered from the 50 kDa fragment and the 100 kDa fragment derived by proteinase K as described in the main text. (B) Escherichia coli harboring plasmid pCJS50( fdeC ) were pulse labeled as in D . Mass spectrometry defined the identity of the 50 kDa N-terminal fragment of FdeC (N), and the 100 kDa C-terminal fragment of FdeC (C).

Techniques Used: Sequencing, Derivative Assay, Plasmid Preparation, Labeling, Mass Spectrometry

7) Product Images from "Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato"

Article Title: Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato

Journal: Frontiers in Plant Science

doi: 10.3389/fpls.2015.01207

Comparison of anti-wilt activity of the fractions obtained by exclusion chromatography of high-molecular weight metabolites from  F. oxysporum  strain CS-20.  Seedlings were inoculated by immersion in a suspension with a final concentration of 10 6  spores/ml, containing spores of two pathogenic strains F37 and Fot3 (1:1). Disease symptoms were examined at 15  (A) , and 21  (B)  days after inoculation of tomato seedlings, which roots were pre-exposed to intact (dark gray columns) or proteinase K-treated (light gray columns) fractions eluted as peaks III, IV, and V. Inoculated seedlings non-exposed to the fractions are referred to as infected control (IfCtrl, black columns). Histograms represent values of average disease index from two independent experiments of each treatment done in triplicate. Bars represent SD.
Figure Legend Snippet: Comparison of anti-wilt activity of the fractions obtained by exclusion chromatography of high-molecular weight metabolites from F. oxysporum strain CS-20. Seedlings were inoculated by immersion in a suspension with a final concentration of 10 6 spores/ml, containing spores of two pathogenic strains F37 and Fot3 (1:1). Disease symptoms were examined at 15 (A) , and 21 (B) days after inoculation of tomato seedlings, which roots were pre-exposed to intact (dark gray columns) or proteinase K-treated (light gray columns) fractions eluted as peaks III, IV, and V. Inoculated seedlings non-exposed to the fractions are referred to as infected control (IfCtrl, black columns). Histograms represent values of average disease index from two independent experiments of each treatment done in triplicate. Bars represent SD.

Techniques Used: Activity Assay, Chromatography, Molecular Weight, Concentration Assay, Infection

Alkalinization of the incubation medium by cultured tomato cells in response to CS-20 proteins contained in fraction V, which reduced Fusarium wilt severity in tomato seedlings. (A) Profiles of extracellular pH change in suspensions of two cell lines after addition of the lyophilized fraction V to a final concentration of 10 μg/ml. Cell response (line β) to the fraction pretreated with proteinase K shown by dotted line. The solid line shows extracellular pH of non-treated cells. (B) Dose-rate effect of the fraction V isolated from CS-20 on tomato cell line γ. Numerals near curves show final concentrations (μg per ml) of the lyophilized fraction V in the tomato cell suspension. Re-stimulation of the reversible response is exemplified for a concentration of 2.5 μg/ml. Dotted line illustrates the irreversibility of alkalinization response to preparation from the pathogenic FOL (strain F37), obtained by the same procedures that were applied to isolate fraction V from CS-20. (C) The extracellular pH values after addition of inactive fractions III (white circles) or IV (white triangles) at a concentration of 10 μg/ml. Asterisks show extracellular pH of non-treated cells. Arrows indicated starting point of treatments. The representative data are out of one of three experiments with the protein fraction V samples independently isolated from CS-20 culture.
Figure Legend Snippet: Alkalinization of the incubation medium by cultured tomato cells in response to CS-20 proteins contained in fraction V, which reduced Fusarium wilt severity in tomato seedlings. (A) Profiles of extracellular pH change in suspensions of two cell lines after addition of the lyophilized fraction V to a final concentration of 10 μg/ml. Cell response (line β) to the fraction pretreated with proteinase K shown by dotted line. The solid line shows extracellular pH of non-treated cells. (B) Dose-rate effect of the fraction V isolated from CS-20 on tomato cell line γ. Numerals near curves show final concentrations (μg per ml) of the lyophilized fraction V in the tomato cell suspension. Re-stimulation of the reversible response is exemplified for a concentration of 2.5 μg/ml. Dotted line illustrates the irreversibility of alkalinization response to preparation from the pathogenic FOL (strain F37), obtained by the same procedures that were applied to isolate fraction V from CS-20. (C) The extracellular pH values after addition of inactive fractions III (white circles) or IV (white triangles) at a concentration of 10 μg/ml. Asterisks show extracellular pH of non-treated cells. Arrows indicated starting point of treatments. The representative data are out of one of three experiments with the protein fraction V samples independently isolated from CS-20 culture.

Techniques Used: Incubation, Cell Culture, Concentration Assay, Isolation

8) Product Images from "The Immunomodulatory Activity of Meningococcal Lipoprotein Ag473 Depends on the Conformation Made up of the Lipid and Protein Moieties"

Article Title: The Immunomodulatory Activity of Meningococcal Lipoprotein Ag473 Depends on the Conformation Made up of the Lipid and Protein Moieties

Journal: PLoS ONE

doi: 10.1371/journal.pone.0040873

The biological activity of L-Ag473 is retained after pretreatment with PMB but abolished by heat or proteinase K treatment. BMDCs were treated with L-Ag473 (100 ng/ml) or LPS (20 ng/ml) for 6 hours and the intracellular TNF-α of CD11c + cells were determined by flow cytometry. Dotted and grey lines represent the isotype-matched control Ab and untreated cell, respectively. PNaseK, proteinase K.
Figure Legend Snippet: The biological activity of L-Ag473 is retained after pretreatment with PMB but abolished by heat or proteinase K treatment. BMDCs were treated with L-Ag473 (100 ng/ml) or LPS (20 ng/ml) for 6 hours and the intracellular TNF-α of CD11c + cells were determined by flow cytometry. Dotted and grey lines represent the isotype-matched control Ab and untreated cell, respectively. PNaseK, proteinase K.

Techniques Used: Activity Assay, Flow Cytometry, Cytometry

The effect of L-Ag473 on human cells. (A) THP-1 cells (2×10 5 /well) were incubated with the indicated amounts of L-Ag473, proteinase K-treated (PNaseK), heated (100°C) L-Ag473 (5 µg/ml). (B) THP-1 cells (2×10 5 /well) were incubated with L-Ag473 (1.5 µg/ml), Pam 3 SK 4  (100 ng/ml) (InvitroGen, Cat No. tlr1-pms) or LPS (100 ng/ml) (Sigma, Cat No. L4391) alone (−PMB) or pretreated with PMB (+PMB) for 18 hours. For antibody blocking experiments, THP-1 cells were preincubated with the indicated antibody for 30 min before stimulation. IL-8 was undetectable in the untreated cells (−). (C) PBMCs (1×10 6 /well) were incubated with the indicated reagent. TNF-α in the 5-h and 24-hours culture supernatants were determined by ELISA. The result shown is the reading values with the untreated cells as the blank. (D) MoDCs were untreated as control, or incubated with L-Ag473 or NL-Ag473 mutant (100 ng/ml) for 16 hours, and then stained with PE-anti-CD86, FITC-anti-CD80, and PE-anti-CD83 mAbs. The MFI is shown on each picture. The data are representative of three independent experiments.
Figure Legend Snippet: The effect of L-Ag473 on human cells. (A) THP-1 cells (2×10 5 /well) were incubated with the indicated amounts of L-Ag473, proteinase K-treated (PNaseK), heated (100°C) L-Ag473 (5 µg/ml). (B) THP-1 cells (2×10 5 /well) were incubated with L-Ag473 (1.5 µg/ml), Pam 3 SK 4 (100 ng/ml) (InvitroGen, Cat No. tlr1-pms) or LPS (100 ng/ml) (Sigma, Cat No. L4391) alone (−PMB) or pretreated with PMB (+PMB) for 18 hours. For antibody blocking experiments, THP-1 cells were preincubated with the indicated antibody for 30 min before stimulation. IL-8 was undetectable in the untreated cells (−). (C) PBMCs (1×10 6 /well) were incubated with the indicated reagent. TNF-α in the 5-h and 24-hours culture supernatants were determined by ELISA. The result shown is the reading values with the untreated cells as the blank. (D) MoDCs were untreated as control, or incubated with L-Ag473 or NL-Ag473 mutant (100 ng/ml) for 16 hours, and then stained with PE-anti-CD86, FITC-anti-CD80, and PE-anti-CD83 mAbs. The MFI is shown on each picture. The data are representative of three independent experiments.

Techniques Used: Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Mutagenesis, Staining

9) Product Images from "Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4"

Article Title: Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20102660

Gliadin-induced innate immune responses are elicited by wheat ATI, a protein copurifying with ω-gliadins. (A) Stimulation of THP-1 cells with α-, γ-, ω1.2-, and ω5-gliadin fractions (all 100 µg/ml) isolated from the pure wheat strain Rektor. Co-incubation of α- and γ-gliadin with 100 µg/ml of regular PT gliadin from Sigma-Aldrich served as cell viability control. LPS was used as positive control, whereas PT or PT zein served as negative control. (B and C) IL-8 secretion after stimulation with 100 µg/ml ω-gliadins in TLR4-transfected (B) and in untransfected HEK-293 cells (C). 10 ng/ml PMA served as cell viability control. 10 ng/ml LPS, 100 µg/ml PT gliadin, or 100 µg/ml of a PT digest of Rektor gliadin (PT Rektor) served as positive control, and 100 µg/ml PT zein, 1 µg/ml Pam3CSK4, or a PT mixture (PT ctrl) served as negative controls. (D) Stimulatory capacity of synthetic overlapping 20mers of ω5-gliadin in TLR4-transfected HEK-293 cells. For illustration purposes, 9 fractions each were pooled in the stimulation experiments (each fraction at a concentration of 100 µg/ml), while also all 43 fractions were tested individually. LPS served as positive and Pam3CSK4 or PT zein as negative controls. (E and F) Dose response of IL-8 release by monocyte-derived DCs stimulated with water-soluble (ws) gliadin (which is enriched in ATI; E) or with purified ATI (F). LPS and water-soluble zein served as positive and negative controls, respectively. (G) Secretion of IL-12 in monocyte-derived DCs from healthy subjects upon stimulation with ATI and PT gliadin in the presence of 1,000 U/ml Interferon-γ as co-stimulatory protein. LPS and PT zein served as positive and negative controls, respectively. (H) Effect of proteinase K digestion of ATI and LPS on IL-8 secretion in DCs. (I) KC secretion in peritoneal macrophages isolated from MyD88 −/− mice compared with C57BL/6J wild-type mice upon ATI or water-soluble gliadin stimulation. LPS and water-soluble zein served as positive and negative controls, respectively. (J) IL-8 secretion of monocyte-derived DCs stimulated with ATI and LPS after preincubation with anti-TLR4 or anti-CD14 antibodies. TLR2 agonist Pam3CSK4 served as positive control. *, P
Figure Legend Snippet: Gliadin-induced innate immune responses are elicited by wheat ATI, a protein copurifying with ω-gliadins. (A) Stimulation of THP-1 cells with α-, γ-, ω1.2-, and ω5-gliadin fractions (all 100 µg/ml) isolated from the pure wheat strain Rektor. Co-incubation of α- and γ-gliadin with 100 µg/ml of regular PT gliadin from Sigma-Aldrich served as cell viability control. LPS was used as positive control, whereas PT or PT zein served as negative control. (B and C) IL-8 secretion after stimulation with 100 µg/ml ω-gliadins in TLR4-transfected (B) and in untransfected HEK-293 cells (C). 10 ng/ml PMA served as cell viability control. 10 ng/ml LPS, 100 µg/ml PT gliadin, or 100 µg/ml of a PT digest of Rektor gliadin (PT Rektor) served as positive control, and 100 µg/ml PT zein, 1 µg/ml Pam3CSK4, or a PT mixture (PT ctrl) served as negative controls. (D) Stimulatory capacity of synthetic overlapping 20mers of ω5-gliadin in TLR4-transfected HEK-293 cells. For illustration purposes, 9 fractions each were pooled in the stimulation experiments (each fraction at a concentration of 100 µg/ml), while also all 43 fractions were tested individually. LPS served as positive and Pam3CSK4 or PT zein as negative controls. (E and F) Dose response of IL-8 release by monocyte-derived DCs stimulated with water-soluble (ws) gliadin (which is enriched in ATI; E) or with purified ATI (F). LPS and water-soluble zein served as positive and negative controls, respectively. (G) Secretion of IL-12 in monocyte-derived DCs from healthy subjects upon stimulation with ATI and PT gliadin in the presence of 1,000 U/ml Interferon-γ as co-stimulatory protein. LPS and PT zein served as positive and negative controls, respectively. (H) Effect of proteinase K digestion of ATI and LPS on IL-8 secretion in DCs. (I) KC secretion in peritoneal macrophages isolated from MyD88 −/− mice compared with C57BL/6J wild-type mice upon ATI or water-soluble gliadin stimulation. LPS and water-soluble zein served as positive and negative controls, respectively. (J) IL-8 secretion of monocyte-derived DCs stimulated with ATI and LPS after preincubation with anti-TLR4 or anti-CD14 antibodies. TLR2 agonist Pam3CSK4 served as positive control. *, P

Techniques Used: Isolation, Incubation, Positive Control, Negative Control, Transfection, Concentration Assay, Derivative Assay, Purification, Mouse Assay

Gliadin leads to secretion of inflammatory cytokines. (A and B) THP-1 cells were treated with PT gliadin, and IL-8 (A) and MCP-1 (B) secretion was measured. LPS and PT zein served as positive and negative controls, respectively. (C) Proteinase K digests of PT gliadin, TNF, and LPS were added to THP-1 cells, and IL-8 secretion was used as read out of innate activation. (D) PT gliadin stimulation of monocyte-derived DCs of healthy controls ( n = 10) and of patients with celiac disease on a gluten-free (gfd; n = 8) or regular diet ( n = 3). Graph shows one representative patient per group. (E) DCs of healthy controls were stimulated with PT gliadin and subjected to analysis by flow cytometry. The dark gray and dotted histograms represent PT gliadin and LPS stimulation, respectively, the light gray histograms show PT zein stimulation, and the dashed lines represent the unstimulated control. (F) HT29, U937, and THP-1 cells were stimulated with gliadin peptide p31-43 or a scrambled control peptide, and IL-8 secretion was measured. *, P
Figure Legend Snippet: Gliadin leads to secretion of inflammatory cytokines. (A and B) THP-1 cells were treated with PT gliadin, and IL-8 (A) and MCP-1 (B) secretion was measured. LPS and PT zein served as positive and negative controls, respectively. (C) Proteinase K digests of PT gliadin, TNF, and LPS were added to THP-1 cells, and IL-8 secretion was used as read out of innate activation. (D) PT gliadin stimulation of monocyte-derived DCs of healthy controls ( n = 10) and of patients with celiac disease on a gluten-free (gfd; n = 8) or regular diet ( n = 3). Graph shows one representative patient per group. (E) DCs of healthy controls were stimulated with PT gliadin and subjected to analysis by flow cytometry. The dark gray and dotted histograms represent PT gliadin and LPS stimulation, respectively, the light gray histograms show PT zein stimulation, and the dashed lines represent the unstimulated control. (F) HT29, U937, and THP-1 cells were stimulated with gliadin peptide p31-43 or a scrambled control peptide, and IL-8 secretion was measured. *, P

Techniques Used: Activation Assay, Derivative Assay, Flow Cytometry, Cytometry

10) Product Images from "Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey"

Article Title: Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey

Journal: Scientific Reports

doi: 10.1038/s41598-018-27449-3

Detection of glucose oxidase (GOX) and defensin-1 (Def-1) in honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following proteinase K treatment by immunoblotting. Aliquots (15 μl) of 50% (w/v) honey solution with or without proteinase K were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 16.5% Tricine-SDS-PAGE. After semi-dry blotting procedure, the blocked membrane was incubated overnight with a rabbit polyclonal antibody against honeybee GOX or Def-1 (1:2000). Shown are cropped blots. (The blots with indicated cropping lines are shown in Supplementary Fig. S2 ). Immunoreactive bands were detected in solution containing dissolved SigmaFast 3,3-diaminobenzidine tablets (GOX) or detected using an enhanced chemiluminescence Immobilon Western kit (Def-1).
Figure Legend Snippet: Detection of glucose oxidase (GOX) and defensin-1 (Def-1) in honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following proteinase K treatment by immunoblotting. Aliquots (15 μl) of 50% (w/v) honey solution with or without proteinase K were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and 16.5% Tricine-SDS-PAGE. After semi-dry blotting procedure, the blocked membrane was incubated overnight with a rabbit polyclonal antibody against honeybee GOX or Def-1 (1:2000). Shown are cropped blots. (The blots with indicated cropping lines are shown in Supplementary Fig. S2 ). Immunoreactive bands were detected in solution containing dissolved SigmaFast 3,3-diaminobenzidine tablets (GOX) or detected using an enhanced chemiluminescence Immobilon Western kit (Def-1).

Techniques Used: Polyacrylamide Gel Electrophoresis, SDS Page, Semi Dry Blot, Incubation, Western Blot

Antibacterial activity of honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following catalase and proteinase K treatment against ( A ) Staphylococcus aureus and ( B ) Pseudomonas aeruginosa isolates. The 50% (w/v) honey solutions were treated with catalase (2000–5000 U/mg protein) at a final concentration ranging from 1000 to 2500 U/ml at room temperature for 2 h or proteinase K (30 U/mg) at a final concentration of 50 μg/ml at 37 °C for 30 min. The antibacterial activity was determined with a minimum inhibitory concentration (MIC) assay. The MIC was defined as the lowest concentration of honey solution (%) inhibiting bacterial growth. K, kanuka honey; M, manuka honey.
Figure Legend Snippet: Antibacterial activity of honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following catalase and proteinase K treatment against ( A ) Staphylococcus aureus and ( B ) Pseudomonas aeruginosa isolates. The 50% (w/v) honey solutions were treated with catalase (2000–5000 U/mg protein) at a final concentration ranging from 1000 to 2500 U/ml at room temperature for 2 h or proteinase K (30 U/mg) at a final concentration of 50 μg/ml at 37 °C for 30 min. The antibacterial activity was determined with a minimum inhibitory concentration (MIC) assay. The MIC was defined as the lowest concentration of honey solution (%) inhibiting bacterial growth. K, kanuka honey; M, manuka honey.

Techniques Used: Activity Assay, Concentration Assay

11) Product Images from "Reconstruction of fish allergenicity from the content and structural traits of the component β-parvalbumin isoforms"

Article Title: Reconstruction of fish allergenicity from the content and structural traits of the component β-parvalbumin isoforms

Journal: Scientific Reports

doi: 10.1038/s41598-019-52801-6

Conformational properties of the Ca 2+ -bound globular folds of the distinct β-PV isoforms. ( a ) DLS analysis of the association state and hydrodynamic features. ( b ) Secondary structure probed by far-UV CD spectra. Spectra were recorded before (circles) and after heating (triangles) at 95 °C. ( c ) Thermal denaturation monitored changes in θ 222 as a function of temperature. Symbol key: •, gmPV1; ◦, gmPV2; , sjPV1; , sjPV2. ( d ) SDS-PAGE analysis of proteinase K and pepsin digestion products of β-PV isoforms. Digestions were performed using a 1/50 protease/β-PV ratio. The full-length gel is displayed in supplementary Fig. 4S .
Figure Legend Snippet: Conformational properties of the Ca 2+ -bound globular folds of the distinct β-PV isoforms. ( a ) DLS analysis of the association state and hydrodynamic features. ( b ) Secondary structure probed by far-UV CD spectra. Spectra were recorded before (circles) and after heating (triangles) at 95 °C. ( c ) Thermal denaturation monitored changes in θ 222 as a function of temperature. Symbol key: •, gmPV1; ◦, gmPV2; , sjPV1; , sjPV2. ( d ) SDS-PAGE analysis of proteinase K and pepsin digestion products of β-PV isoforms. Digestions were performed using a 1/50 protease/β-PV ratio. The full-length gel is displayed in supplementary Fig. 4S .

Techniques Used: SDS Page

Features of the amyloid aggregates formed by β-PV isoforms. ( a ) Far-UV CD spectra of the insoluble aggregates of gmPV1, gmPV2 and sjPV2. ( b ) Dot blot analysis of (A) β-PV aggregates using amyloid-specific anti-fibril (OC) and anti-oligomer (A11) antibodies. (G) Ca 2+ -bound chains were included as controls. ( c ) Coomassie blue-stained SDS-PAGE gel of the harsh proteinase K and pepsin digestions of gmPV1, gmPV2 and sjPV2 amyloid aggregates. The original membranes and gels of panels b and c are displayed in supplementary Fig. 6S .
Figure Legend Snippet: Features of the amyloid aggregates formed by β-PV isoforms. ( a ) Far-UV CD spectra of the insoluble aggregates of gmPV1, gmPV2 and sjPV2. ( b ) Dot blot analysis of (A) β-PV aggregates using amyloid-specific anti-fibril (OC) and anti-oligomer (A11) antibodies. (G) Ca 2+ -bound chains were included as controls. ( c ) Coomassie blue-stained SDS-PAGE gel of the harsh proteinase K and pepsin digestions of gmPV1, gmPV2 and sjPV2 amyloid aggregates. The original membranes and gels of panels b and c are displayed in supplementary Fig. 6S .

Techniques Used: Dot Blot, Staining, SDS Page

12) Product Images from "Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications"

Article Title: Minimizing carry-over PCR contamination in expanded CAG/CTG repeat instability applications

Journal: Scientific Reports

doi: 10.1038/s41598-017-18168-2

Using heat-labile Ung and/or Proteinase K prevents the degradation of uracil-containing amplicons. ( A ) Experimental scheme used. ( B ) Agarose gel of amplicons from genomic DNA of GFP(CAG) 101 containing uracil and treated post-PCR with (lanes 7–11) or without (lanes 2–6) proteinase K three days after the PCR was performed (lanes 3–6 and 8–11) or immediately after the PCR (fresh, lanes 2, 7). The samples were stored at −20 °C (lanes 3, 8), 4 °C (lanes 4, 9), room temperature (RT°, lanes 5, 10), or were amplified using a heat labile (hl) Ung instead of the conventional Ung and stored at room temperature (lanes 6,11). MW = molecular weight marker (lanes 1 and 12).
Figure Legend Snippet: Using heat-labile Ung and/or Proteinase K prevents the degradation of uracil-containing amplicons. ( A ) Experimental scheme used. ( B ) Agarose gel of amplicons from genomic DNA of GFP(CAG) 101 containing uracil and treated post-PCR with (lanes 7–11) or without (lanes 2–6) proteinase K three days after the PCR was performed (lanes 3–6 and 8–11) or immediately after the PCR (fresh, lanes 2, 7). The samples were stored at −20 °C (lanes 3, 8), 4 °C (lanes 4, 9), room temperature (RT°, lanes 5, 10), or were amplified using a heat labile (hl) Ung instead of the conventional Ung and stored at room temperature (lanes 6,11). MW = molecular weight marker (lanes 1 and 12).

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

Small-pool PCR of expanded repeats using Ung and dUTP. ( A ) SP-PCR blot from reactions pretreated with the standard Ung, amplified with dUTP, and stopped using Proteinase K. The visible bands are shorter than in the samples seen in B., and the smearing below the bands is evident. Each lane contains the product of a PCR with 50 pg of template DNA derived from GFP(CAG) 101  treated with sgCTG and the Cas9 nickase. ( B ) SP-PCR blots of amplicons generated using dTTP or dUTP, pretreated with hl Ung, and stopped after the PCR with Proteinase K. Each lane contains the product of a PCR that used 250 pg of genomic DNA from GFP(CAG) 101  treated with the Cas9 nickase and either the sgCTG or an empty guide RNA vector (pPN10). In all cases, the probe used was generated with dUTP.
Figure Legend Snippet: Small-pool PCR of expanded repeats using Ung and dUTP. ( A ) SP-PCR blot from reactions pretreated with the standard Ung, amplified with dUTP, and stopped using Proteinase K. The visible bands are shorter than in the samples seen in B., and the smearing below the bands is evident. Each lane contains the product of a PCR with 50 pg of template DNA derived from GFP(CAG) 101 treated with sgCTG and the Cas9 nickase. ( B ) SP-PCR blots of amplicons generated using dTTP or dUTP, pretreated with hl Ung, and stopped after the PCR with Proteinase K. Each lane contains the product of a PCR that used 250 pg of genomic DNA from GFP(CAG) 101 treated with the Cas9 nickase and either the sgCTG or an empty guide RNA vector (pPN10). In all cases, the probe used was generated with dUTP.

Techniques Used: Polymerase Chain Reaction, Amplification, Derivative Assay, Generated, Plasmid Preparation

13) Product Images from "The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω-Hexatoxin-Hv1a"

Article Title: The Cystine Knot Is Responsible for the Exceptional Stability of the Insecticidal Spider Toxin ω-Hexatoxin-Hv1a

Journal: Toxins

doi: 10.3390/toxins7104366

Inhibitor cystine knot of the spider-venom peptide ω-hexatoxin-Hv1a. ( A ) Schematic of the ICK motif, which comprises an antiparallel β sheet stabilised by a cystine knot [ 1 ]. β strands are shown in green and the six cysteine residues that form the cystine knot are labeled 1–6. In spider toxins, the β sheet typically comprises only the two β strands housing Cys5 and Cys6, although a third N -terminal strand encompassing Cys2 is sometimes present [ 3 ]. The two “outer” disulfide bonds are shown in green and the “inner” disulfide bridge is red. ( B ) Schematic of the three-dimensional structure of the 37-residue spider-venom peptide Hv1a (PDB 1AXH) [ 5 ] highlighting the ICK motif. The cystine knot comprises a ring formed by two disulfides (Cys1–Cys4 and Cys2–Cys5, green) and the intervening sections of polypeptide backbone (gray), with a third disulfide (Cys3–Cys6, red) piercing the ring to create a pseudo-knot. The hydrophobic core of the toxin consists primarily of the two central disulfide bridges connected to the β strands. ( C ) Primary structure of Hv1a showing the location of the three disulfide bonds and the 10 proteinase K cleavage sites predicted by PeptideCutter [ 6 ].
Figure Legend Snippet: Inhibitor cystine knot of the spider-venom peptide ω-hexatoxin-Hv1a. ( A ) Schematic of the ICK motif, which comprises an antiparallel β sheet stabilised by a cystine knot [ 1 ]. β strands are shown in green and the six cysteine residues that form the cystine knot are labeled 1–6. In spider toxins, the β sheet typically comprises only the two β strands housing Cys5 and Cys6, although a third N -terminal strand encompassing Cys2 is sometimes present [ 3 ]. The two “outer” disulfide bonds are shown in green and the “inner” disulfide bridge is red. ( B ) Schematic of the three-dimensional structure of the 37-residue spider-venom peptide Hv1a (PDB 1AXH) [ 5 ] highlighting the ICK motif. The cystine knot comprises a ring formed by two disulfides (Cys1–Cys4 and Cys2–Cys5, green) and the intervening sections of polypeptide backbone (gray), with a third disulfide (Cys3–Cys6, red) piercing the ring to create a pseudo-knot. The hydrophobic core of the toxin consists primarily of the two central disulfide bridges connected to the β strands. ( C ) Primary structure of Hv1a showing the location of the three disulfide bonds and the 10 proteinase K cleavage sites predicted by PeptideCutter [ 6 ].

Techniques Used: Labeling

Proteolytic stability of Hv1a. Fraction of intact native Hv1a (black circles) and linear Hv1a (grey squares) after incubation with proteinase K (1:200 molar ratio) at pH 7.5 and 37 °C for up to 24 h. The values for native Hv1a were quantified relative to a sample of native Hv1a incubated for 24 h under the same conditions without proteinase K. Values for linear Hv1a were quantified relative to a sample of linear Hv1a at pH 7.5 incubated for 0 h without proteinase K. The dashed line indicates 50% intact toxin. Data are mean ± SD.
Figure Legend Snippet: Proteolytic stability of Hv1a. Fraction of intact native Hv1a (black circles) and linear Hv1a (grey squares) after incubation with proteinase K (1:200 molar ratio) at pH 7.5 and 37 °C for up to 24 h. The values for native Hv1a were quantified relative to a sample of native Hv1a incubated for 24 h under the same conditions without proteinase K. Values for linear Hv1a were quantified relative to a sample of linear Hv1a at pH 7.5 incubated for 0 h without proteinase K. The dashed line indicates 50% intact toxin. Data are mean ± SD.

Techniques Used: Incubation

14) Product Images from "Chlamydial Lipoproteins Stimulate Toll-Like Receptors 1/2 Mediated Inflammatory Responses through MyD88-Dependent Pathway"

Article Title: Chlamydial Lipoproteins Stimulate Toll-Like Receptors 1/2 Mediated Inflammatory Responses through MyD88-Dependent Pathway

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00078

Production of proinflammatory cytokines induced by D775 required lipid modification while D541 did not . TLR4 −/− mouse macrophage were incubated with lipoproteins treated with lipase, lipoproteins treated with proteinase, lipase alone, or proteinase alone. Lipoprotein+lipase groups: D775 and D541 were incubated with1000 U/ml pig pancreas lipase at 37°C for 16 h, followed by heating at 100°C before beginning cytokine stimulation assay. Lipoprotein+ proteinase groups: D775 or D541 with 100 μg/ml proteinase K at 37°C for 2 h, followed by addition of 200 mM PMSF. Lipoprotein groups: D775 and D541 were incubated in enzyme free buffer as controls. Cell-free supernatants were harvested after 4 h incubation for cytokine mMIP-2 measurement by ELISA. It confirmed that lipid modification of D775 was essential for its stimulation of proinflammatory cytokine production, but D541 stimulation was not greatly affected by lipase.
Figure Legend Snippet: Production of proinflammatory cytokines induced by D775 required lipid modification while D541 did not . TLR4 −/− mouse macrophage were incubated with lipoproteins treated with lipase, lipoproteins treated with proteinase, lipase alone, or proteinase alone. Lipoprotein+lipase groups: D775 and D541 were incubated with1000 U/ml pig pancreas lipase at 37°C for 16 h, followed by heating at 100°C before beginning cytokine stimulation assay. Lipoprotein+ proteinase groups: D775 or D541 with 100 μg/ml proteinase K at 37°C for 2 h, followed by addition of 200 mM PMSF. Lipoprotein groups: D775 and D541 were incubated in enzyme free buffer as controls. Cell-free supernatants were harvested after 4 h incubation for cytokine mMIP-2 measurement by ELISA. It confirmed that lipid modification of D775 was essential for its stimulation of proinflammatory cytokine production, but D541 stimulation was not greatly affected by lipase.

Techniques Used: Modification, Incubation, Enzyme-linked Immunosorbent Assay

15) Product Images from "Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes"

Article Title: Cell-Free Phospholipid Biosynthesis by Gene-Encoded Enzymes Reconstituted in Liposomes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0163058

Synthesis of 18:1 LPA and DOPA from GPAT and LPAAT enzymes produced inside liposomes. ( a ) Schematic of vesicle-confined experiments. PURE frex supplemented with the plsB and plsC genes and with 500 μM G3P was encapsulated inside liposomes using gentle rehydration of a lipid film covering sub-millimetre glass beads. Lipid composition consisted of DOPC, DOPE, DOPG, cardiolipin, TexasRed-DHPE and DSPE-PEG-biotin (Table B in S1 File ). Swelling occurred at 4°C to avoid reaction initiation. Gene expression outside liposomes was inhibited by protein digestion. Lipid biosynthesis was triggered by external supply of 100 μM oleoyl-CoA (o-CoA). ( b ) Confocal microscopy images of liposomes after swelling. Vesicles were labelled with a membrane dye (Texas-Red). Scale bar is 5 μm. ( c , d ) Concentration of 18:1 LPA ( c ) and DOPA ( d ) synthesized in a one-pot reaction by GPAT and LPAAT enzymes produced outside liposomes composed of DOPG, DOPE and cardiolipin (Table B in S1 File ). Lipid precursors were 500 μM G3P and 100 μM o-CoA (except in negative control). Error bars indicate s.e.m. of two injections of the same sample. ( e , f ) Concentration of 18:1 LPA ( e ) and DOPA ( f ) produced by GPAT and LPAAT enzymes generated inside liposomes. Three experimental configurations corresponding to different localizations of protein digestion were tested. As expected, addition of Proteinase K both inside and outside liposomes totally inhibited lipid synthesis. In the absence of Proteinase K 18:1 LPA and DOPA accumulated as a result of both internal and external acyltransferase production. Liposome-confined IVTT and lipid synthesis was demonstrated by supplementing Proteinase K outside vesicles according to the reaction scheme illustrated in (a). Data are mean and s.e.m. of three independent experiments. For each replicate the same sample was injected two times in the MS, their averaged value was calculated and data are reported as the mean and standard error across the three trials.
Figure Legend Snippet: Synthesis of 18:1 LPA and DOPA from GPAT and LPAAT enzymes produced inside liposomes. ( a ) Schematic of vesicle-confined experiments. PURE frex supplemented with the plsB and plsC genes and with 500 μM G3P was encapsulated inside liposomes using gentle rehydration of a lipid film covering sub-millimetre glass beads. Lipid composition consisted of DOPC, DOPE, DOPG, cardiolipin, TexasRed-DHPE and DSPE-PEG-biotin (Table B in S1 File ). Swelling occurred at 4°C to avoid reaction initiation. Gene expression outside liposomes was inhibited by protein digestion. Lipid biosynthesis was triggered by external supply of 100 μM oleoyl-CoA (o-CoA). ( b ) Confocal microscopy images of liposomes after swelling. Vesicles were labelled with a membrane dye (Texas-Red). Scale bar is 5 μm. ( c , d ) Concentration of 18:1 LPA ( c ) and DOPA ( d ) synthesized in a one-pot reaction by GPAT and LPAAT enzymes produced outside liposomes composed of DOPG, DOPE and cardiolipin (Table B in S1 File ). Lipid precursors were 500 μM G3P and 100 μM o-CoA (except in negative control). Error bars indicate s.e.m. of two injections of the same sample. ( e , f ) Concentration of 18:1 LPA ( e ) and DOPA ( f ) produced by GPAT and LPAAT enzymes generated inside liposomes. Three experimental configurations corresponding to different localizations of protein digestion were tested. As expected, addition of Proteinase K both inside and outside liposomes totally inhibited lipid synthesis. In the absence of Proteinase K 18:1 LPA and DOPA accumulated as a result of both internal and external acyltransferase production. Liposome-confined IVTT and lipid synthesis was demonstrated by supplementing Proteinase K outside vesicles according to the reaction scheme illustrated in (a). Data are mean and s.e.m. of three independent experiments. For each replicate the same sample was injected two times in the MS, their averaged value was calculated and data are reported as the mean and standard error across the three trials.

Techniques Used: Produced, Expressing, Confocal Microscopy, Concentration Assay, Synthesized, Negative Control, Generated, Injection, Mass Spectrometry

16) Product Images from "Effect of Divalent Metals on the Neuronal Proteasomal System, Prion Protein Ubiquitination and Aggregation"

Article Title: Effect of Divalent Metals on the Neuronal Proteasomal System, Prion Protein Ubiquitination and Aggregation

Journal: Toxicology letters

doi: 10.1016/j.toxlet.2012.09.008

3.4. Cadmium induces formation of soluble oligomers, but not Proteinase-K resistant prion proteins
Figure Legend Snippet: 3.4. Cadmium induces formation of soluble oligomers, but not Proteinase-K resistant prion proteins

Techniques Used:

In vitro cadmium treatment does not alter PrPC susceptibility to proteinase-K digestion
Figure Legend Snippet: In vitro cadmium treatment does not alter PrPC susceptibility to proteinase-K digestion

Techniques Used: In Vitro

17) Product Images from "BAC Transgenic Mice Expressing a Truncated Mutant Parkin Exhibit Age-dependent Hypokinetic Motor Deficits, Dopaminergic Neuron Degeneration, and Accumulation of Proteinase K-Resistant Alpha-Synuclein"

Article Title: BAC Transgenic Mice Expressing a Truncated Mutant Parkin Exhibit Age-dependent Hypokinetic Motor Deficits, Dopaminergic Neuron Degeneration, and Accumulation of Proteinase K-Resistant Alpha-Synuclein

Journal: The Journal of neuroscience : the official journal of the Society for Neuroscience

doi: 10.1523/JNEUROSCI.5351-08.2009

Accumulation of proteinase K resistant alpha-synuclein in nigra of Parkin-Q311X(A) mice. A - B , Photomicrographs of the midbrain sections of Parkin-Q311X(A) mice and wild-type littermates at 3 months of age immunostained for proteinase-K resistant alpha-synuclein
Figure Legend Snippet: Accumulation of proteinase K resistant alpha-synuclein in nigra of Parkin-Q311X(A) mice. A - B , Photomicrographs of the midbrain sections of Parkin-Q311X(A) mice and wild-type littermates at 3 months of age immunostained for proteinase-K resistant alpha-synuclein

Techniques Used: Mouse Assay

Colocalization of nitrotyrosine and proteinase K resistant alpha-synuclein in nigra of the Parkin-Q311X(A) mice. Confocal microphotographs of double immunofluroresence labelling proteinase K resistant alpha-synuclein (green) and 3-NT (red) in nigra of
Figure Legend Snippet: Colocalization of nitrotyrosine and proteinase K resistant alpha-synuclein in nigra of the Parkin-Q311X(A) mice. Confocal microphotographs of double immunofluroresence labelling proteinase K resistant alpha-synuclein (green) and 3-NT (red) in nigra of

Techniques Used: Mouse Assay

18) Product Images from "Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection"

Article Title: Type I Interferon Production Induced by Streptococcus pyogenes-Derived Nucleic Acids Is Required for Host Protection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1001345

S. pyogenes -derived RNA and DNA delivered to endosomes induce IFN-β in cDCs and BMDMs, respectively. S. pyogenes  cells were sonicated and the extracts were treated with either DNase I, RNase A, Proteinase K, or left untreated (control extract). The extracts and a reagent control were delivered into cDCs derived from TLR7 -/-  ( A ), MyD88 -/-  ( B ) and MAVS -/-  ( C ) or control (WT) mice using DOTAP. After stimulation for 6 h, supernatants were collected and IFN-β release was measured using ELISA. Values represent mean ± SD; n = 3. ( D ) BMDMs from TLR9 -/-  and control (WT) mice were transfected with streptococcal extracts as described in (A). After 8 h supernatants were collected for IFN-β measurements by ELISA. ( E)  Purified RNA (5 µg) from  S. pyogenes  (SP), Group B streptococcus (GBS),  Staphylococcus aureus  (SA),  Listeria monocytogenes  (LM) and RAW 264.7 cells (RAW) were delivered into cDCs using DOTAP. After stimulation for 6 h supernatants were collected and IFN-β release was measured. Values represent mean ± SD; n = 3. ( F ) DNA (5 µg) from same organisms as in (E) as well as poly(dA:dT) were delivered into BMDMs and 8 h later IFN-β release was determined as in (E).
Figure Legend Snippet: S. pyogenes -derived RNA and DNA delivered to endosomes induce IFN-β in cDCs and BMDMs, respectively. S. pyogenes cells were sonicated and the extracts were treated with either DNase I, RNase A, Proteinase K, or left untreated (control extract). The extracts and a reagent control were delivered into cDCs derived from TLR7 -/- ( A ), MyD88 -/- ( B ) and MAVS -/- ( C ) or control (WT) mice using DOTAP. After stimulation for 6 h, supernatants were collected and IFN-β release was measured using ELISA. Values represent mean ± SD; n = 3. ( D ) BMDMs from TLR9 -/- and control (WT) mice were transfected with streptococcal extracts as described in (A). After 8 h supernatants were collected for IFN-β measurements by ELISA. ( E) Purified RNA (5 µg) from S. pyogenes (SP), Group B streptococcus (GBS), Staphylococcus aureus (SA), Listeria monocytogenes (LM) and RAW 264.7 cells (RAW) were delivered into cDCs using DOTAP. After stimulation for 6 h supernatants were collected and IFN-β release was measured. Values represent mean ± SD; n = 3. ( F ) DNA (5 µg) from same organisms as in (E) as well as poly(dA:dT) were delivered into BMDMs and 8 h later IFN-β release was determined as in (E).

Techniques Used: Derivative Assay, Sonication, Mouse Assay, Enzyme-linked Immunosorbent Assay, Transfection, Purification

19) Product Images from "A simple in vitro assay for assessing the efficacy, mechanisms and kinetics of anti-prion fibril compounds"

Article Title: A simple in vitro assay for assessing the efficacy, mechanisms and kinetics of anti-prion fibril compounds

Journal: Prion

doi: 10.1080/19336896.2018.1525254

Cell-based assay was used to identify anti-amyloid small molecules that have anti-prion activity. Western blot of PrP with and without proteinase K (PK) digestion, from scrapie infected neuroblastoma cells (ScN2A) that were incubated with pterostilbene, trans-resveratrol, rosmarinic acid, myricetin, and ferulic acid, at 100 and 50 µM (panel a). The blank had the same volume of dimethyl sulfoxide (DMSO) added as the drug tests. The effect of four concentrations of rosmarinic acid and myricetin (25, 50, 100 and 200 µM) on PK resistance in ScN2A was used to determine the IC 50 (panel b). The gel used to generate the PK digested panels in panel B is shown in Suppl. Figure 6.
Figure Legend Snippet: Cell-based assay was used to identify anti-amyloid small molecules that have anti-prion activity. Western blot of PrP with and without proteinase K (PK) digestion, from scrapie infected neuroblastoma cells (ScN2A) that were incubated with pterostilbene, trans-resveratrol, rosmarinic acid, myricetin, and ferulic acid, at 100 and 50 µM (panel a). The blank had the same volume of dimethyl sulfoxide (DMSO) added as the drug tests. The effect of four concentrations of rosmarinic acid and myricetin (25, 50, 100 and 200 µM) on PK resistance in ScN2A was used to determine the IC 50 (panel b). The gel used to generate the PK digested panels in panel B is shown in Suppl. Figure 6.

Techniques Used: Cell Based Assay, Activity Assay, Western Blot, Infection, Incubation

20) Product Images from "Suppression of αvβ6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells"

Article Title: Suppression of αvβ6 Integrin Expression by Polymicrobial Oral Biofilms in Gingival Epithelial Cells

Journal: Scientific Reports

doi: 10.1038/s41598-017-03619-7

The role of TLR ligands in the downregulation of β6 integrin in GECs. ( A ) and ( B ) GECs were treated with FSL-1 (100 ng/ml; n = 4), Pam3CSK4 (300 ng/ml; n = 4), flagellin (100 ng/ml; n = 3), LTA (2 µg/ml; n = 3) or MALP-2 (100 ng/ml; n = 3) or left untreated for 32 h, and their effect on ITGB6 ( A ) and IL1B expression ( B ) was analyzed by RT-qPCR. Mean ± SEM is presented. ( C ), GECs were pre-treated with anti-TLR2 and anti-TLR6 blocking antibodies for 1 h, after which heated biofilm extract (60 µg protein/ml) or FSL-1 (100 ng/ml) was added for 32 h. ITGB6 expression was analyzed by RT-qPCR. Mean ± SEM is presented (n = 3–5). ( D ) GECs were treated with heated Mycoplasma salivarium extract (10 7 or 10 8 cfu/ml) with or without proteinase K digestion for 32 h and their ITGB6 expression was analyzed by RT-qPCR. Heated biofilm extract (60 µg protein/ml) and FSL-1 (100 ng/ml) were used as positive controls. Mean ± SEM of three experiments is presented. *p
Figure Legend Snippet: The role of TLR ligands in the downregulation of β6 integrin in GECs. ( A ) and ( B ) GECs were treated with FSL-1 (100 ng/ml; n = 4), Pam3CSK4 (300 ng/ml; n = 4), flagellin (100 ng/ml; n = 3), LTA (2 µg/ml; n = 3) or MALP-2 (100 ng/ml; n = 3) or left untreated for 32 h, and their effect on ITGB6 ( A ) and IL1B expression ( B ) was analyzed by RT-qPCR. Mean ± SEM is presented. ( C ), GECs were pre-treated with anti-TLR2 and anti-TLR6 blocking antibodies for 1 h, after which heated biofilm extract (60 µg protein/ml) or FSL-1 (100 ng/ml) was added for 32 h. ITGB6 expression was analyzed by RT-qPCR. Mean ± SEM is presented (n = 3–5). ( D ) GECs were treated with heated Mycoplasma salivarium extract (10 7 or 10 8 cfu/ml) with or without proteinase K digestion for 32 h and their ITGB6 expression was analyzed by RT-qPCR. Heated biofilm extract (60 µg protein/ml) and FSL-1 (100 ng/ml) were used as positive controls. Mean ± SEM of three experiments is presented. *p

Techniques Used: Expressing, Quantitative RT-PCR, Blocking Assay

21) Product Images from "A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in Neural Progenitor Cells"

Article Title: A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in Neural Progenitor Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0072039

Dextran-FITC diffusion in brain specimens. 50 µm slices from a 4 mm hemisphere “stained” with dextran-FITC for 24 hours under different conditions as shown. Average one-dimensional intensity profiles from the edge of the tissue toward the center across several samples are shown at lower right with 90% confidence intervals. Samples processed for different lengths of time were combined by scaling the linear diffusion distance by the square root of time. The combination of increasing temperature, utilizing a 1% PFA fixation, and processing with a freeze/thaw, and proteinase K digestion (+FT) resulted in the biggest improvement to penetration.
Figure Legend Snippet: Dextran-FITC diffusion in brain specimens. 50 µm slices from a 4 mm hemisphere “stained” with dextran-FITC for 24 hours under different conditions as shown. Average one-dimensional intensity profiles from the edge of the tissue toward the center across several samples are shown at lower right with 90% confidence intervals. Samples processed for different lengths of time were combined by scaling the linear diffusion distance by the square root of time. The combination of increasing temperature, utilizing a 1% PFA fixation, and processing with a freeze/thaw, and proteinase K digestion (+FT) resulted in the biggest improvement to penetration.

Techniques Used: Diffusion-based Assay

22) Product Images from "Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence"

Article Title: Mycobacterial tlyA gene product is localized to the cell-wall without signal sequence

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2015.00060

Susceptibility to extrinsic proteases of TlyA expressing  M. smegmatis  (A,B) and  E. coli  (C,D): M. smegmatis /TlyA  (C)  and  E. coli /TlyA  (D)  were treated with Proteinase K and the resultant samples were processed for 12% SDS–PAGE and detected with anti-TlyA antibody. P and S indicate pellet and supernatant obtained at indicated times in minutes. The antibodies used for the detection of the blot are indicated on the right side. The lane marked with TlyA indicates purified TlyA as control. The  (B,D)  respectively represent the hemolytic activity of the Proteinase K treated bacteria, shown in  (A , C ). The labels are indicated below the bars. Proteinase K treated bacteria.(7 × 10 7 ) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis by measuring the absorbance at 540 nm of RBC free supernatant.
Figure Legend Snippet: Susceptibility to extrinsic proteases of TlyA expressing M. smegmatis (A,B) and E. coli (C,D): M. smegmatis /TlyA (C) and E. coli /TlyA (D) were treated with Proteinase K and the resultant samples were processed for 12% SDS–PAGE and detected with anti-TlyA antibody. P and S indicate pellet and supernatant obtained at indicated times in minutes. The antibodies used for the detection of the blot are indicated on the right side. The lane marked with TlyA indicates purified TlyA as control. The (B,D) respectively represent the hemolytic activity of the Proteinase K treated bacteria, shown in (A , C ). The labels are indicated below the bars. Proteinase K treated bacteria.(7 × 10 7 ) were incubated with 1% rabbit RBC at room temp for 24 h to assess the degree of lysis by measuring the absorbance at 540 nm of RBC free supernatant.

Techniques Used: Expressing, SDS Page, Purification, Activity Assay, Incubation, Lysis

23) Product Images from "The effect of truncation on prion-like properties of α-synuclein"

Article Title: The effect of truncation on prion-like properties of α-synuclein

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.001862

Concentration-dependent proteinase K digestion assays of αS aggregates. αS fibrils were treated with PK (0, 1, 10, 100 μg/ml) for 30 min at 37 °C. After treatment, the samples were resolved using SDS-PAGE and stained by CBB. A , FL and truncated human αS fibril seeds. B , FL mouse αS aggregates seeded with mouse WT αS fibril seeds (WT Mo ), human WT αS fibril seeds (WT Hu ), and human truncated αS fibril seeds. A filled circle indicates the band at ∼12 kDa; a filled arrowhead indicates the band at ∼10 kDa; an open arrowhead indicates the band at ∼8 kDa. Three independent experiments were done and representative data are shown.
Figure Legend Snippet: Concentration-dependent proteinase K digestion assays of αS aggregates. αS fibrils were treated with PK (0, 1, 10, 100 μg/ml) for 30 min at 37 °C. After treatment, the samples were resolved using SDS-PAGE and stained by CBB. A , FL and truncated human αS fibril seeds. B , FL mouse αS aggregates seeded with mouse WT αS fibril seeds (WT Mo ), human WT αS fibril seeds (WT Hu ), and human truncated αS fibril seeds. A filled circle indicates the band at ∼12 kDa; a filled arrowhead indicates the band at ∼10 kDa; an open arrowhead indicates the band at ∼8 kDa. Three independent experiments were done and representative data are shown.

Techniques Used: Concentration Assay, SDS Page, Staining

24) Product Images from "TANGO-Inspired Design of Anti-Amyloid Cyclic Peptides"

Article Title: TANGO-Inspired Design of Anti-Amyloid Cyclic Peptides

Journal: ACS chemical neuroscience

doi: 10.1021/acschemneuro.6b00150

Effect of CG3 and CG8 on A β fibril formation investigated by proteolytic fragmentation assay. A β samples were incubated alone or with mTTR, CG3, or CG8 for 24 h. (a) Relative rate of proteolytic fragmentation of A β was measured by subsequent addition of proteinase K followed by dotting onto nitrocellulose membrane at different time points. Unfragmented A β species were detected by 6E10 antibody. (b) Density of dots was quantified using ImageJ. The data were fit to a single exponential decay and half-times were determined to be 38 min for A β , 18 min for A β +CG3, and 10 min for A β +CG8 and A β +mTTR.
Figure Legend Snippet: Effect of CG3 and CG8 on A β fibril formation investigated by proteolytic fragmentation assay. A β samples were incubated alone or with mTTR, CG3, or CG8 for 24 h. (a) Relative rate of proteolytic fragmentation of A β was measured by subsequent addition of proteinase K followed by dotting onto nitrocellulose membrane at different time points. Unfragmented A β species were detected by 6E10 antibody. (b) Density of dots was quantified using ImageJ. The data were fit to a single exponential decay and half-times were determined to be 38 min for A β , 18 min for A β +CG3, and 10 min for A β +CG8 and A β +mTTR.

Techniques Used: Incubation, Relative Rate

25) Product Images from "Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage"

Article Title: Isolation and molecular characterisation of Achromobacter phage phiAxp-3, an N4-like bacteriophage

Journal: Scientific Reports

doi: 10.1038/srep24776

The effects of various bacterial treatments on phiAxp-3 adsorption to host cells, as determined by residual plaque-forming unit percentages. ( a ) Effect of proteinase K treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. ( b ) Effect of periodate treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732+IO 4− ” for periodate treatment) groups were tested for adsorption as indicated by the x axes. Error bars denote statistical variations. Statistical significance was determined by a Student t test for comparison between the treated and untreated groups. * P 0.05. ( c ) Inactivation of phage phiAxp-3 by lipopolysaccharide derived from A. xylosoxidans A22732. The percentage infectivity was determined after 1 h of incubation at 37 °C. Error bars denote statistical variations.
Figure Legend Snippet: The effects of various bacterial treatments on phiAxp-3 adsorption to host cells, as determined by residual plaque-forming unit percentages. ( a ) Effect of proteinase K treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. ( b ) Effect of periodate treatment on the adsorption of phiAxp-3 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732+IO 4− ” for periodate treatment) groups were tested for adsorption as indicated by the x axes. Error bars denote statistical variations. Statistical significance was determined by a Student t test for comparison between the treated and untreated groups. * P 0.05. ( c ) Inactivation of phage phiAxp-3 by lipopolysaccharide derived from A. xylosoxidans A22732. The percentage infectivity was determined after 1 h of incubation at 37 °C. Error bars denote statistical variations.

Techniques Used: Adsorption, Derivative Assay, Infection, Incubation

26) Product Images from "Characterization of a novel Achromobacter xylosoxidans specific siphoviruse: phiAxp-1"

Article Title: Characterization of a novel Achromobacter xylosoxidans specific siphoviruse: phiAxp-1

Journal: Scientific Reports

doi: 10.1038/srep21943

Effects of different treatments of bacteria on phiAxp-1 adsorption. The results are shown as residual PFU percentages. ( A ) Effect of proteinase K treatment on the adsorption of phiAxp-1 to A. xylosoxidans strain A22732. ( B ) Effect of periodate treatment on the adsorption of phiAxp-1 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732 + IO 4− ” for periodate treatment) groups were tested for adsorption, as indicated in the x- axes. Error bars denote statistical variations. Significance was determined by a Student’s t test for comparison between the treated and the untreated groups. * P 0.05.
Figure Legend Snippet: Effects of different treatments of bacteria on phiAxp-1 adsorption. The results are shown as residual PFU percentages. ( A ) Effect of proteinase K treatment on the adsorption of phiAxp-1 to A. xylosoxidans strain A22732. ( B ) Effect of periodate treatment on the adsorption of phiAxp-1 to A. xylosoxidans strain A22732. The control (LB and “A22732 + acetate”), untreated strain (A22732), and treatment (“A22732 + ProtK” for proteinase K treatment and “A22732 + IO 4− ” for periodate treatment) groups were tested for adsorption, as indicated in the x- axes. Error bars denote statistical variations. Significance was determined by a Student’s t test for comparison between the treated and the untreated groups. * P 0.05.

Techniques Used: Adsorption

27) Product Images from "Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast"

Article Title: Din7 and Mhr1 expression levels regulate double-strand-break-induced replication and recombination of mtDNA at ori5 in yeast

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt273

Overproduction of Din7 enhances 5′−3′ dsDNA exonuclease activity in mitochondria. ( A ) Principle of the 5′−3′ and 3′−5′ exonuclease activity assay. The 5′- and 3′- 32 P-labels were preferentially removed by 5′−3′ and 3′−5′ exonuclease activity, and they are resistant to 3′−5′ and 5′−3′ exonucleases, respectively. ( B ) Schematic diagram of the construction of the Din7 deletion mutant (Din7Δ). Immunoblot analysis of overproduced C-terminally 6× His-tagged Din7 or Din7Δ in mitochondrial extracts (bottom). Mitochondria were isolated from cells bearing WT/pYES2/CT, WT/pYES2/CT-DIN7 and WT/pYES2/CT-din7Δ (overproducing Din7Δ with a C-terminal 6× His tag). The signals of overproduced Din7 and Din7Δ were detected using a monoclonal anti-6× His-tag antibody. As a control, the levels of porin (a constitutively expressed protein, used here as a control) were determined by immunoblot analysis using a monoclonal anti-porin antibody to adjust the protein concentrations of mitochondrial extracts from all three strains to the same level. ( C and E ) Analyses of 5′−3′ or 3′−5′ exonuclease activity in mitochondrial extracts. The 5′- or 3′- 32 P-labeled pUC119/HincII dsDNA fragments were incubated at 37°C for 15 min with increasing concentrations of mitochondrial extracts. After removing the proteins by treating the extracts with proteinase K, the DNA molecules were separated on a 1% agarose gel. ( D and F ) Quantitative representation of the 5′−3′ and 3′−5′ exonuclease activities in mitochondrial extracts, detected in (C) and (E). Signals relative to untreated DNA are plotted. ds, double-stranded; ss, single-stranded. Open circles, incubated with mitochondrial extracts from wild-type cells (cells without Din7 overproduction); closed circles, incubated with mitochondrial extracts from Din7-overproducing cells; closed triangles, incubated with mitochondrial extracts from Din7Δ-overproducing cells. Each bar represents the results of at least two independent experiments. ( G ) The generation of single-stranded DNA by Din7. HincII-linearized pUC119 DNA (104 µM) was treated at 37°C for 5 min with increasing amounts of mitochondrial extracts derived from cells of WT/pYES2/CT, WT/pYES2/CT-DIN7 or WT/pYES2/CT-din7Δ in the same buffer used for detection of λ exonuclease activity. Then, each reaction solution (10 µl) was separated into duplicate aliquots. Proteinase K was added to one aliquot to stop the reaction. The second aliquot from each set was treated at 37°C for 10 min with 2.25 U mung bean nuclease followed by addition of proteinase K to stop the reaction. Mung bean nuclease digests only the single-stranded DNA, leaving the double-stranded DNA intact. Samples were then electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lane 1, 1-kb plus ladder used as a size marker; lane 2, HincII-linearized pUC119 DNA; lane 3, HincII-linearized pUC119 DNA treated in a reaction mixture without mitochondrial extracts; lanes 4–7, HincII-linearized pUC119 DNA treated with increasing amounts of mitochondrial extracts; lane 8, HincII-linearized pUC119 DNA treated only with mung bean nuclease; lanes 9–12, samples treated as in lanes 4–7, treated additionally with mung bean nuclease. The samples treated with mitochondrial extracts overexpressing DIN7 contain both single-stranded DNA fragments with discrete sizes and double-stranded DNA because discrete DNA signals smaller than those of the double-stranded DNA in the middle were removed by mung bean nuclease treatments.
Figure Legend Snippet: Overproduction of Din7 enhances 5′−3′ dsDNA exonuclease activity in mitochondria. ( A ) Principle of the 5′−3′ and 3′−5′ exonuclease activity assay. The 5′- and 3′- 32 P-labels were preferentially removed by 5′−3′ and 3′−5′ exonuclease activity, and they are resistant to 3′−5′ and 5′−3′ exonucleases, respectively. ( B ) Schematic diagram of the construction of the Din7 deletion mutant (Din7Δ). Immunoblot analysis of overproduced C-terminally 6× His-tagged Din7 or Din7Δ in mitochondrial extracts (bottom). Mitochondria were isolated from cells bearing WT/pYES2/CT, WT/pYES2/CT-DIN7 and WT/pYES2/CT-din7Δ (overproducing Din7Δ with a C-terminal 6× His tag). The signals of overproduced Din7 and Din7Δ were detected using a monoclonal anti-6× His-tag antibody. As a control, the levels of porin (a constitutively expressed protein, used here as a control) were determined by immunoblot analysis using a monoclonal anti-porin antibody to adjust the protein concentrations of mitochondrial extracts from all three strains to the same level. ( C and E ) Analyses of 5′−3′ or 3′−5′ exonuclease activity in mitochondrial extracts. The 5′- or 3′- 32 P-labeled pUC119/HincII dsDNA fragments were incubated at 37°C for 15 min with increasing concentrations of mitochondrial extracts. After removing the proteins by treating the extracts with proteinase K, the DNA molecules were separated on a 1% agarose gel. ( D and F ) Quantitative representation of the 5′−3′ and 3′−5′ exonuclease activities in mitochondrial extracts, detected in (C) and (E). Signals relative to untreated DNA are plotted. ds, double-stranded; ss, single-stranded. Open circles, incubated with mitochondrial extracts from wild-type cells (cells without Din7 overproduction); closed circles, incubated with mitochondrial extracts from Din7-overproducing cells; closed triangles, incubated with mitochondrial extracts from Din7Δ-overproducing cells. Each bar represents the results of at least two independent experiments. ( G ) The generation of single-stranded DNA by Din7. HincII-linearized pUC119 DNA (104 µM) was treated at 37°C for 5 min with increasing amounts of mitochondrial extracts derived from cells of WT/pYES2/CT, WT/pYES2/CT-DIN7 or WT/pYES2/CT-din7Δ in the same buffer used for detection of λ exonuclease activity. Then, each reaction solution (10 µl) was separated into duplicate aliquots. Proteinase K was added to one aliquot to stop the reaction. The second aliquot from each set was treated at 37°C for 10 min with 2.25 U mung bean nuclease followed by addition of proteinase K to stop the reaction. Mung bean nuclease digests only the single-stranded DNA, leaving the double-stranded DNA intact. Samples were then electrophoresed on a 1% agarose gel and stained with ethidium bromide. Lane 1, 1-kb plus ladder used as a size marker; lane 2, HincII-linearized pUC119 DNA; lane 3, HincII-linearized pUC119 DNA treated in a reaction mixture without mitochondrial extracts; lanes 4–7, HincII-linearized pUC119 DNA treated with increasing amounts of mitochondrial extracts; lane 8, HincII-linearized pUC119 DNA treated only with mung bean nuclease; lanes 9–12, samples treated as in lanes 4–7, treated additionally with mung bean nuclease. The samples treated with mitochondrial extracts overexpressing DIN7 contain both single-stranded DNA fragments with discrete sizes and double-stranded DNA because discrete DNA signals smaller than those of the double-stranded DNA in the middle were removed by mung bean nuclease treatments.

Techniques Used: Activity Assay, Mutagenesis, Isolation, Labeling, Incubation, Agarose Gel Electrophoresis, Derivative Assay, Staining, Marker

28) Product Images from "Endoplasmic reticulum–associated degradation and disposal of misfolded GPI-anchored proteins in Trypanosoma brucei"

Article Title: Endoplasmic reticulum–associated degradation and disposal of misfolded GPI-anchored proteins in Trypanosoma brucei

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E18-06-0380

Characterization of MG132 protected species. All analyses were performed after RNAi silencing (24 h) of endogenous TfR. All MG132 treatments were for 2 h at 25 μM. (A) HA:E6- and HA:E6ΔG-expressing cells were incubated without (Cont) or with MG132 (MG). The cells were hypotonically lysed and total (T), cytoplasmic (C), and membrane (M) fractions were prepared. All fractions (10 7 cell equivalents/lane) were analyzed by immunoblotting with anti-HA (αHA), anti-Hsp70 (αH70, cytoplasmic marker), or anti-BiP (αBiP, ER marker). (B) Cell fractions prepared from MG132-treated HA:E6 cells were treated with proteinase K (Prot-K) as indicated in the absence or presence of NP40. Samples (10 7 cell equivalents/lane) were immunoblotted with anti-HA, anti-Hsp70, and anti-BiP. (C–E) HA:E6- or HA:E6ΔG-expressing cells were incubated without (-) or with (+) MG132 treatment. (C) Cells were solubilized under denaturing conditions and each was split into two equal fractions (10 7 cell equivalents). One set was mock-treated (-) and the other digested (+) with PNGase F (PNG). Samples were analyzed by immunoblotting with anti-HA. (D) Lysates were immunoprecipitated with anti-TfR antibodies (10 7 cell equivalents/precipitate) covalently cross-linked to Protein A sepharose. One set (right) was immunoblotted with anti-HA (αHA) and the other blotted with biotinylated ConA (left). Mobilities of E6 and molecular mass markers (kDa) are indicated on the left. White strip indicates digital reordering of lanes after image processing. Stars in A–D indicate mobility of MG132-protected E6 or fully deglycosylated species. (E) Total extracts of HA:E6-expressing cells were prepared in SDS sample buffer, and lysates for immunoprecipitation were prepared identically to GPI-PLC-lysates in Figure 8 to minimize deubiquitinating activities. Total cell extracts (lanes 1–4) and anti-HA immunoprecipitates (lanes 5–8) were immunoblotted (10 7 cell equivalents/lane) sequentially with anti-ubiquitin and anti-HA on the same membrane and the individual signals were processed prior to digital separation for presentation.
Figure Legend Snippet: Characterization of MG132 protected species. All analyses were performed after RNAi silencing (24 h) of endogenous TfR. All MG132 treatments were for 2 h at 25 μM. (A) HA:E6- and HA:E6ΔG-expressing cells were incubated without (Cont) or with MG132 (MG). The cells were hypotonically lysed and total (T), cytoplasmic (C), and membrane (M) fractions were prepared. All fractions (10 7 cell equivalents/lane) were analyzed by immunoblotting with anti-HA (αHA), anti-Hsp70 (αH70, cytoplasmic marker), or anti-BiP (αBiP, ER marker). (B) Cell fractions prepared from MG132-treated HA:E6 cells were treated with proteinase K (Prot-K) as indicated in the absence or presence of NP40. Samples (10 7 cell equivalents/lane) were immunoblotted with anti-HA, anti-Hsp70, and anti-BiP. (C–E) HA:E6- or HA:E6ΔG-expressing cells were incubated without (-) or with (+) MG132 treatment. (C) Cells were solubilized under denaturing conditions and each was split into two equal fractions (10 7 cell equivalents). One set was mock-treated (-) and the other digested (+) with PNGase F (PNG). Samples were analyzed by immunoblotting with anti-HA. (D) Lysates were immunoprecipitated with anti-TfR antibodies (10 7 cell equivalents/precipitate) covalently cross-linked to Protein A sepharose. One set (right) was immunoblotted with anti-HA (αHA) and the other blotted with biotinylated ConA (left). Mobilities of E6 and molecular mass markers (kDa) are indicated on the left. White strip indicates digital reordering of lanes after image processing. Stars in A–D indicate mobility of MG132-protected E6 or fully deglycosylated species. (E) Total extracts of HA:E6-expressing cells were prepared in SDS sample buffer, and lysates for immunoprecipitation were prepared identically to GPI-PLC-lysates in Figure 8 to minimize deubiquitinating activities. Total cell extracts (lanes 1–4) and anti-HA immunoprecipitates (lanes 5–8) were immunoblotted (10 7 cell equivalents/lane) sequentially with anti-ubiquitin and anti-HA on the same membrane and the individual signals were processed prior to digital separation for presentation.

Techniques Used: Expressing, Incubation, Marker, Immunoprecipitation, Stripping Membranes, Planar Chromatography

29) Product Images from "Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey"

Article Title: Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey

Journal: Scientific Reports

doi: 10.1038/s41598-018-27449-3

Antibacterial activity of honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following catalase and proteinase K treatment against ( A ) Staphylococcus aureus and ( B ) Pseudomonas aeruginosa isolates. The 50% (w/v) honey solutions were treated with catalase (2000–5000 U/mg protein) at a final concentration ranging from 1000 to 2500 U/ml at room temperature for 2 h or proteinase K (30 U/mg) at a final concentration of 50 μg/ml at 37 °C for 30 min. The antibacterial activity was determined with a minimum inhibitory concentration (MIC) assay. The MIC was defined as the lowest concentration of honey solution (%) inhibiting bacterial growth. K, kanuka honey; M, manuka honey.
Figure Legend Snippet: Antibacterial activity of honeydew honey samples (n = 23) and medical-grade manuka and kanuka honey following catalase and proteinase K treatment against ( A ) Staphylococcus aureus and ( B ) Pseudomonas aeruginosa isolates. The 50% (w/v) honey solutions were treated with catalase (2000–5000 U/mg protein) at a final concentration ranging from 1000 to 2500 U/ml at room temperature for 2 h or proteinase K (30 U/mg) at a final concentration of 50 μg/ml at 37 °C for 30 min. The antibacterial activity was determined with a minimum inhibitory concentration (MIC) assay. The MIC was defined as the lowest concentration of honey solution (%) inhibiting bacterial growth. K, kanuka honey; M, manuka honey.

Techniques Used: Activity Assay, Concentration Assay

30) Product Images from "Extracellular Protease Digestion to Evaluate Membrane Protein Cell Surface Localization"

Article Title: Extracellular Protease Digestion to Evaluate Membrane Protein Cell Surface Localization

Journal: Nature protocols

doi: 10.1038/nprot.2015.131

Western blots showing the resistance of YapV45, MBP and SurA to digestion by proteinase K (proK) or trypsin (Tryp) in intact cells or sonicated cell lysates. (a) Proteinase K treatment of E. coli BL21(DE3)pLysS expressing YapV, YapV45 or harboring the empty vector (EV). YapV is digested on the surface of intact cells. When cells are lysed by sonication prior to proK treatment, the cytoplasm localized YapV45 and the periplasmic chaperone SurA are digested, but the periplasmic MBP is not digested. (b) Trypsin treatment results in the same digestion pattern as proK. (c) Proteinase K treatment of several additional strains of E. coli . MBP is resistant to digestion by proK in all strains tested, even after cell lysis by sonication prior to proK treatment. In contrast, SurA remains undigested after proK treatment of intact cells, but is digested by proK when cells are lysed by sonication prior to proK treatment.
Figure Legend Snippet: Western blots showing the resistance of YapV45, MBP and SurA to digestion by proteinase K (proK) or trypsin (Tryp) in intact cells or sonicated cell lysates. (a) Proteinase K treatment of E. coli BL21(DE3)pLysS expressing YapV, YapV45 or harboring the empty vector (EV). YapV is digested on the surface of intact cells. When cells are lysed by sonication prior to proK treatment, the cytoplasm localized YapV45 and the periplasmic chaperone SurA are digested, but the periplasmic MBP is not digested. (b) Trypsin treatment results in the same digestion pattern as proK. (c) Proteinase K treatment of several additional strains of E. coli . MBP is resistant to digestion by proK in all strains tested, even after cell lysis by sonication prior to proK treatment. In contrast, SurA remains undigested after proK treatment of intact cells, but is digested by proK when cells are lysed by sonication prior to proK treatment.

Techniques Used: Western Blot, Sonication, Expressing, Plasmid Preparation, Lysis

31) Product Images from "The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps"

Article Title: The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps

Journal: PLoS ONE

doi: 10.1371/journal.pone.0048606

ScARPs are surface exposed. (A) Western immunoblot analysis of ScARPs from S. citri GII3. Spiroplasmas were incubated either with the reaction buffer alone (−) or with 1 mg/ml trypsin (+), for 1, 4, and 24 h. Proteins were probed with MAb 10G3. (B–D) Western immunoblot analysis of S. citri GII3 treated by proteinase K during 10 min at various concentrations. Proteins were probed with anti-P32 PAbs (B), MAb 10G3 (C), and anti-Rep3d PAbs (D).
Figure Legend Snippet: ScARPs are surface exposed. (A) Western immunoblot analysis of ScARPs from S. citri GII3. Spiroplasmas were incubated either with the reaction buffer alone (−) or with 1 mg/ml trypsin (+), for 1, 4, and 24 h. Proteins were probed with MAb 10G3. (B–D) Western immunoblot analysis of S. citri GII3 treated by proteinase K during 10 min at various concentrations. Proteins were probed with anti-P32 PAbs (B), MAb 10G3 (C), and anti-Rep3d PAbs (D).

Techniques Used: Western Blot, Incubation

32) Product Images from "The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola"

Article Title: The major outer sheath protein forms distinct conformers and multimeric complexes in the outer membrane and periplasm of Treponema denticola

Journal: Scientific Reports

doi: 10.1038/s41598-017-13550-6

Immunolabeling and surface proteolysis of T. denticola confirm bipartite architecture of native MOSP and the presence of OM and periplasmic conformers. ( A ) Immunolabeling of T. denticola encapsulated in gel microdroplets in the absence (−) or presence (+) of 0.05% TX-100. Organisms were probed with antibodies directed against MOSP C , MOSP N , and periplasmic flagella (PF). ( B ) Surface proteolysis of T. denticola exposed to proteinase K (PK) for 1 hour. Immunoblot analysis of MOSP before (−) and after (+) treatment with PK using antisera directed against MOSP Fl , MOSP C and MOSP N . Molecular mass standards (kDa) are indicated on the left of each gel.
Figure Legend Snippet: Immunolabeling and surface proteolysis of T. denticola confirm bipartite architecture of native MOSP and the presence of OM and periplasmic conformers. ( A ) Immunolabeling of T. denticola encapsulated in gel microdroplets in the absence (−) or presence (+) of 0.05% TX-100. Organisms were probed with antibodies directed against MOSP C , MOSP N , and periplasmic flagella (PF). ( B ) Surface proteolysis of T. denticola exposed to proteinase K (PK) for 1 hour. Immunoblot analysis of MOSP before (−) and after (+) treatment with PK using antisera directed against MOSP Fl , MOSP C and MOSP N . Molecular mass standards (kDa) are indicated on the left of each gel.

Techniques Used: Immunolabeling

33) Product Images from "High Manganese Tolerance and Biooxidation Ability of Serratia marcescens Isolated from Manganese Mine Water in Minas Gerais, Brazil"

Article Title: High Manganese Tolerance and Biooxidation Ability of Serratia marcescens Isolated from Manganese Mine Water in Minas Gerais, Brazil

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.01946

Decay of manganese concentration during manganese removal assay and Mn(II) oxidation in cell-free filtrates of  (A)  CL11 and  (B)  CL35 under standard conditions or after the addition of proteinase K. The isolates were grown in K medium and the supernatant was recovered to evaluate the oxidation of Mn as described in section “Materials and Methods.” Samples were collected periodically and manganese removal was determined by ICP-OES. The presence of manganese oxide was monitored by the addition of LBB to the samples. The error bars indicates the standard deviation of the biological triplicate.
Figure Legend Snippet: Decay of manganese concentration during manganese removal assay and Mn(II) oxidation in cell-free filtrates of (A) CL11 and (B) CL35 under standard conditions or after the addition of proteinase K. The isolates were grown in K medium and the supernatant was recovered to evaluate the oxidation of Mn as described in section “Materials and Methods.” Samples were collected periodically and manganese removal was determined by ICP-OES. The presence of manganese oxide was monitored by the addition of LBB to the samples. The error bars indicates the standard deviation of the biological triplicate.

Techniques Used: Concentration Assay, Standard Deviation

34) Product Images from "Staphylococcal Enterotoxins Dose-Dependently Modulate the Generation of Myeloid-Derived Suppressor Cells"

Article Title: Staphylococcal Enterotoxins Dose-Dependently Modulate the Generation of Myeloid-Derived Suppressor Cells

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00321

PMN-like MDSC levels are modulated by heat-stable S. aureus proteins sized 10 to 50 kDa. S. aureus supernatants were pre-treated prior to stimulation of PBMC. (A) Pre-treatment with 20 μg/ml of proteinase K at 37°C for 16 h, (B) heat-treatment at 80°C for 20 min, (C) filtration through spin columns with MWCO cut-off pores as indicated. 3% (USA300 or Newman) or 10% (NCTC 8325) of supernatants were used as high concentrations (black bars), 0.01% (all tested strains) were used as low concentrations (gray bars). Data are presented as means ± SEM. Differences between stimulated samples and medium controls were analyzed by a one-sample t -test. Differences between different pre-treatment conditions (A,B) were analyzed by a Mann-Whitney test. * p
Figure Legend Snippet: PMN-like MDSC levels are modulated by heat-stable S. aureus proteins sized 10 to 50 kDa. S. aureus supernatants were pre-treated prior to stimulation of PBMC. (A) Pre-treatment with 20 μg/ml of proteinase K at 37°C for 16 h, (B) heat-treatment at 80°C for 20 min, (C) filtration through spin columns with MWCO cut-off pores as indicated. 3% (USA300 or Newman) or 10% (NCTC 8325) of supernatants were used as high concentrations (black bars), 0.01% (all tested strains) were used as low concentrations (gray bars). Data are presented as means ± SEM. Differences between stimulated samples and medium controls were analyzed by a one-sample t -test. Differences between different pre-treatment conditions (A,B) were analyzed by a Mann-Whitney test. * p

Techniques Used: Filtration, MANN-WHITNEY

35) Product Images from "Staphylococcal Enterotoxins Dose-Dependently Modulate the Generation of Myeloid-Derived Suppressor Cells"

Article Title: Staphylococcal Enterotoxins Dose-Dependently Modulate the Generation of Myeloid-Derived Suppressor Cells

Journal: Frontiers in Cellular and Infection Microbiology

doi: 10.3389/fcimb.2018.00321

PMN-like MDSC levels are modulated by heat-stable S. aureus proteins sized 10 to 50 kDa. S. aureus supernatants were pre-treated prior to stimulation of PBMC. (A) Pre-treatment with 20 μg/ml of proteinase K at 37°C for 16 h, (B) heat-treatment at 80°C for 20 min, (C) filtration through spin columns with MWCO cut-off pores as indicated. 3% (USA300 or Newman) or 10% (NCTC 8325) of supernatants were used as high concentrations (black bars), 0.01% (all tested strains) were used as low concentrations (gray bars). Data are presented as means ± SEM. Differences between stimulated samples and medium controls were analyzed by a one-sample t -test. Differences between different pre-treatment conditions (A,B) were analyzed by a Mann-Whitney test. * p
Figure Legend Snippet: PMN-like MDSC levels are modulated by heat-stable S. aureus proteins sized 10 to 50 kDa. S. aureus supernatants were pre-treated prior to stimulation of PBMC. (A) Pre-treatment with 20 μg/ml of proteinase K at 37°C for 16 h, (B) heat-treatment at 80°C for 20 min, (C) filtration through spin columns with MWCO cut-off pores as indicated. 3% (USA300 or Newman) or 10% (NCTC 8325) of supernatants were used as high concentrations (black bars), 0.01% (all tested strains) were used as low concentrations (gray bars). Data are presented as means ± SEM. Differences between stimulated samples and medium controls were analyzed by a one-sample t -test. Differences between different pre-treatment conditions (A,B) were analyzed by a Mann-Whitney test. * p

Techniques Used: Filtration, MANN-WHITNEY

36) Product Images from "Outer Membrane Proteins Ail and OmpF of Yersinia pestis Are Involved in the Adsorption of T7-Related Bacteriophage Yep-phi"

Article Title: Outer Membrane Proteins Ail and OmpF of Yersinia pestis Are Involved in the Adsorption of T7-Related Bacteriophage Yep-phi

Journal: Journal of Virology

doi: 10.1128/JVI.01948-13

Effects of the different treatments of bacteria on Yep-phi adsorption, which are shown as residual PFU percentages. (A) Effect of proteinase K treatment on the adsorption of Yep-phi to Yersinia pestis strain 201. (B) Effect of periodate treatment on the
Figure Legend Snippet: Effects of the different treatments of bacteria on Yep-phi adsorption, which are shown as residual PFU percentages. (A) Effect of proteinase K treatment on the adsorption of Yep-phi to Yersinia pestis strain 201. (B) Effect of periodate treatment on the

Techniques Used: Adsorption

37) Product Images from "Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans"

Article Title: Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans

Journal: Scientific Reports

doi: 10.1038/srep34300

Effects of different treatments applied to the host bacterium on phiAxp-2 adsorption, which is shown as residual pfu percentages. ( a ) Effect of proteinase K treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. ( b ) Effect of periodate treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. The controls (LB and “A22732+ acetate”), untreated (A22732), and treated groups (“A22732+ ProtK”, treated with proteinase K; “A22732+ IO 4− ”, treated with periodate) were tested for adsorption, as indicated on the x axes. Error bars denote statistical variations. Significance was determined with one-sample Student’s t test when the treated and untreated groups were compared. * P
Figure Legend Snippet: Effects of different treatments applied to the host bacterium on phiAxp-2 adsorption, which is shown as residual pfu percentages. ( a ) Effect of proteinase K treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. ( b ) Effect of periodate treatment on the adsorption of phiAxp-2 to A. xylosoxidans strain A22732. The controls (LB and “A22732+ acetate”), untreated (A22732), and treated groups (“A22732+ ProtK”, treated with proteinase K; “A22732+ IO 4− ”, treated with periodate) were tested for adsorption, as indicated on the x axes. Error bars denote statistical variations. Significance was determined with one-sample Student’s t test when the treated and untreated groups were compared. * P

Techniques Used: Adsorption

38) Product Images from "Tenfibgen Ligand Nanoencapsulation Delivers Bi-Functional Anti-CK2 RNAi Oligomer to Key Sites for Prostate Cancer Targeting Using Human Xenograft Tumors in Mice"

Article Title: Tenfibgen Ligand Nanoencapsulation Delivers Bi-Functional Anti-CK2 RNAi Oligomer to Key Sites for Prostate Cancer Targeting Using Human Xenograft Tumors in Mice

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109970

Nanocapsule design, morphology, cargo stability, and cargo protection. ( a ) Cartoon depiction of nanocapsule design. ( b ) Transmission electron micrograph of s50-TBG-RNAi-CK2 nanocapsules for in vivo studies. Magnification 230,000×. Scale bar 100 nm. ( c ) Left panel: Naked RNAi-CK2 oligomer was digested with proteinase K for 24 to 96 h. Inp, undigested input oligomer. Right panel: Naked and s50-TBG encapsulated RNAi-CK2 oligomers were digested with DNase followed by proteinase K as indicated above the panel. Lanes 1 2, naked RNAi-CK2; 3 4, naked RNAi-CK2 with TBG-sugar nanocapsules included in the digestion; 5 - 7, in vitro use formulation of s50-TBG-RNAi-CK2; 8 – 10, in vivo use formulation of s50-TBG-RNAi-CK2.
Figure Legend Snippet: Nanocapsule design, morphology, cargo stability, and cargo protection. ( a ) Cartoon depiction of nanocapsule design. ( b ) Transmission electron micrograph of s50-TBG-RNAi-CK2 nanocapsules for in vivo studies. Magnification 230,000×. Scale bar 100 nm. ( c ) Left panel: Naked RNAi-CK2 oligomer was digested with proteinase K for 24 to 96 h. Inp, undigested input oligomer. Right panel: Naked and s50-TBG encapsulated RNAi-CK2 oligomers were digested with DNase followed by proteinase K as indicated above the panel. Lanes 1 2, naked RNAi-CK2; 3 4, naked RNAi-CK2 with TBG-sugar nanocapsules included in the digestion; 5 - 7, in vitro use formulation of s50-TBG-RNAi-CK2; 8 – 10, in vivo use formulation of s50-TBG-RNAi-CK2.

Techniques Used: Transmission Assay, In Vivo, In Vitro

39) Product Images from "Dissection of the Kaposi's Sarcoma-Associated Herpesvirus Gene Expression Program by Using the Viral DNA Replication Inhibitor Cidofovir"

Article Title: Dissection of the Kaposi's Sarcoma-Associated Herpesvirus Gene Expression Program by Using the Viral DNA Replication Inhibitor Cidofovir

Journal: Journal of Virology

doi: 10.1128/JVI.78.24.13637-13652.2004

The effect of CDV on KSHV RNA synthesis, cell viability, and virion production. (A) Real-time RT-PCR assay for KSHV ORF 17 RNA. RNA was harvested at 72 h after the induction of lytic replication from BCBL-1 cells pretreated with 0, 10, 25, 50, 75, and 100 μM CDV. The TPA-induced and CDV-treated ORF 17 cycle threshold (Ct) values were normalized against endogenous GAPDH Ct (dCt) values, and the Ct value for the latent background non-CDV-treated samples was subtracted to determine the net expression (ddCt). Data values were calculated as the amplification efficiency of the ORF 17 probe, one doubling per cycle, raised to the power of −ddCT (2 −ddCt ) to determine the change ( n -fold) in expression. Data values from three independent experiments are plotted as the average percentages for the maximal TPA-induced, non-CDV-treated (0 μM) conditions set at 100% expression. (B) BCBL-1 cells with 100 μM CDV pretreatment (indicated with a white X on a black circle and a stippled line) and non-CDV-treated cells (black diamond with solid line) were induced with 20 ng of TPA/ml and grown in parallel with untreated cells (open triangle with dashed lines). Samples were collected at serial time points, and viability was determined with trypan blue staining. Data shown are the averages of three separate experiments. The presence of CDV appeared to have no effect on BCBL-1 cell viability. (C) Real-time PCR assay for virion DNA. Supernatant was collected at the indicated time points from untreated (open bar), TPA-induced (black bar), and TPA-induced and 100 μM CDV-treated (gray-shaded bar) cell culture samples and assayed for KSHV DNA after an initial DNase treatment, followed by SDS-proteinase K treatment and DNA extraction. CDV suppresses late KSHV replication in a dose-dependent manner and inhibits free virion DNA to near-baseline levels.
Figure Legend Snippet: The effect of CDV on KSHV RNA synthesis, cell viability, and virion production. (A) Real-time RT-PCR assay for KSHV ORF 17 RNA. RNA was harvested at 72 h after the induction of lytic replication from BCBL-1 cells pretreated with 0, 10, 25, 50, 75, and 100 μM CDV. The TPA-induced and CDV-treated ORF 17 cycle threshold (Ct) values were normalized against endogenous GAPDH Ct (dCt) values, and the Ct value for the latent background non-CDV-treated samples was subtracted to determine the net expression (ddCt). Data values were calculated as the amplification efficiency of the ORF 17 probe, one doubling per cycle, raised to the power of −ddCT (2 −ddCt ) to determine the change ( n -fold) in expression. Data values from three independent experiments are plotted as the average percentages for the maximal TPA-induced, non-CDV-treated (0 μM) conditions set at 100% expression. (B) BCBL-1 cells with 100 μM CDV pretreatment (indicated with a white X on a black circle and a stippled line) and non-CDV-treated cells (black diamond with solid line) were induced with 20 ng of TPA/ml and grown in parallel with untreated cells (open triangle with dashed lines). Samples were collected at serial time points, and viability was determined with trypan blue staining. Data shown are the averages of three separate experiments. The presence of CDV appeared to have no effect on BCBL-1 cell viability. (C) Real-time PCR assay for virion DNA. Supernatant was collected at the indicated time points from untreated (open bar), TPA-induced (black bar), and TPA-induced and 100 μM CDV-treated (gray-shaded bar) cell culture samples and assayed for KSHV DNA after an initial DNase treatment, followed by SDS-proteinase K treatment and DNA extraction. CDV suppresses late KSHV replication in a dose-dependent manner and inhibits free virion DNA to near-baseline levels.

Techniques Used: Quantitative RT-PCR, Expressing, Amplification, Staining, Real-time Polymerase Chain Reaction, Cell Culture, DNA Extraction

40) Product Images from "Determination of the membrane topology of lemur tyrosine kinase 2 (LMTK2) by fluorescence protease protection"

Article Title: Determination of the membrane topology of lemur tyrosine kinase 2 (LMTK2) by fluorescence protease protection

Journal: American Journal of Physiology - Cell Physiology

doi: 10.1152/ajpcell.00288.2012

Biochemical protease protection assay reveals cytoplasmic NH 2 - or COOH termini. Hek293 cells expressing NH 2 - or COOH-terminal tagged LMTK2 were digitonin permeabilized followed by incubation ± proteinase K. Cells were lysed and proteins resolved
Figure Legend Snippet: Biochemical protease protection assay reveals cytoplasmic NH 2 - or COOH termini. Hek293 cells expressing NH 2 - or COOH-terminal tagged LMTK2 were digitonin permeabilized followed by incubation ± proteinase K. Cells were lysed and proteins resolved

Techniques Used: Expressing, Incubation

41) Product Images from "Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to ?-Sheet Rich Oligomers and Fibrils"

Article Title: Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to ?-Sheet Rich Oligomers and Fibrils

Journal: PLoS ONE

doi: 10.1371/journal.pone.0098753

Shaking-induced fibrils have Proteinase K resistance. SDS-PAGE of recMoPrP c 23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.
Figure Legend Snippet: Shaking-induced fibrils have Proteinase K resistance. SDS-PAGE of recMoPrP c 23–231 (panel A) and fibrils (panel B) without (PK-) and with PK at 1∶50, 1∶200 and 1∶400 (PK:PrP, g:g) shows that shaking-induced fibrils have 12, 13, 14 and 17 kDa resistance bands.

Techniques Used: SDS Page

42) Product Images from "TANGO-Inspired Design of Anti-Amyloid Cyclic Peptides"

Article Title: TANGO-Inspired Design of Anti-Amyloid Cyclic Peptides

Journal: ACS chemical neuroscience

doi: 10.1021/acschemneuro.6b00150

Effect of CG3 and CG8 on A β fibril formation investigated by proteolytic fragmentation assay. A β samples were incubated alone or with mTTR, CG3, or CG8 for 24 h. (a) Relative rate of proteolytic fragmentation of A β was measured by subsequent addition of proteinase K followed by dotting onto nitrocellulose membrane at different time points. Unfragmented A β species were detected by 6E10 antibody. (b) Density of dots was quantified using ImageJ. The data were fit to a single exponential decay and half-times were determined to be 38 min for A β , 18 min for A β +CG3, and 10 min for A β +CG8 and A β +mTTR.
Figure Legend Snippet: Effect of CG3 and CG8 on A β fibril formation investigated by proteolytic fragmentation assay. A β samples were incubated alone or with mTTR, CG3, or CG8 for 24 h. (a) Relative rate of proteolytic fragmentation of A β was measured by subsequent addition of proteinase K followed by dotting onto nitrocellulose membrane at different time points. Unfragmented A β species were detected by 6E10 antibody. (b) Density of dots was quantified using ImageJ. The data were fit to a single exponential decay and half-times were determined to be 38 min for A β , 18 min for A β +CG3, and 10 min for A β +CG8 and A β +mTTR.

Techniques Used: Incubation, Relative Rate

43) Product Images from "Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ?A1122 ▿"

Article Title: Identification of the Lipopolysaccharide Core of Yersinia pestis and Yersinia pseudotuberculosis as the Receptor for Bacteriophage ?A1122 ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00339-11

Effects of different treatments of bacteria on φA1122 adsorption, shown as residual PFU percentages. (A) The effect of proteinase K treatment on adsorption of φA1122 to Y. pestis and Y. pseudotuberculosis. (B) The effect of periodate treatment
Figure Legend Snippet: Effects of different treatments of bacteria on φA1122 adsorption, shown as residual PFU percentages. (A) The effect of proteinase K treatment on adsorption of φA1122 to Y. pestis and Y. pseudotuberculosis. (B) The effect of periodate treatment

Techniques Used: Adsorption

44) Product Images from "Development of a keratinase activity assay using recombinant chicken feather keratin substrates"

Article Title: Development of a keratinase activity assay using recombinant chicken feather keratin substrates

Journal: PLoS ONE

doi: 10.1371/journal.pone.0172712

The degree of keratin degradation by AWCE. (A) Purification of fluorescein-5-maleimide (FM)-conjugated Chr27_FK12 β-keratin by Superdex 200 10/300 GL column chromatography. (B) Relative degradation of FM-conjugated Chr27_FK12 by AWCE and proteinase K.
Figure Legend Snippet: The degree of keratin degradation by AWCE. (A) Purification of fluorescein-5-maleimide (FM)-conjugated Chr27_FK12 β-keratin by Superdex 200 10/300 GL column chromatography. (B) Relative degradation of FM-conjugated Chr27_FK12 by AWCE and proteinase K.

Techniques Used: Purification, Column Chromatography

45) Product Images from "Ovulation-inducing factor: a protein component of llama seminal plasma"

Article Title: Ovulation-inducing factor: a protein component of llama seminal plasma

Journal: Reproductive Biology and Endocrinology : RB & E

doi: 10.1186/1477-7827-8-44

Llama seminal plasma protein separation on SDS-PAGE after exposure to different treatments in Experiment 2 (A) and Experiment 3 (B) . Seminal plasma samples were reduced, denatured, and separated on 12% polyacrylamide gel and stained with Coomassie Blue R-250.  A)  Lane 1 was loaded with a molecular mass standard. The remaining lanes were loaded with 20 μg of seminal plasma (SP): Lane 2 - non-treated SP; Lane 3 - SP kept at 38°C for 1 hour; Lane 4 - SP heated to 65°C for 10 min.; Lane 5 - SP heated to 65°C for 1 hour; Lane 6 -- SP treated with charcoal dextran at 4°C for 12 hours; Lane 7 -- SP treated with proteinase K (500 μg/ml) at 38°C for 1 hour; Lane 8 -- phosphate buffered saline (PBS) plus proteinase K (500 μg/ml) at 38°C for 1 hour; * Protein band represents proteinase K.  B)  Lane 1 was loaded with a molecular mass standard. The remaining lanes were loaded with 30 μg of seminal plasma: Lane 2 - non-treated seminal plasma (SP); Lane 3 - SP kept at 38°C for 12 hours; Lane 4 - phosphate buffered saline (PBS) plus pronase E (500 μg/ml) at 38°C for 1 hour; Lane 5, 6, 7, 8, 9 - Seminal plasma treated with pronase E (500 μg/ml) at 38°C for 1, 3, 6, 9 or 12 hours, respectively. * Protein bands represent pronase E.
Figure Legend Snippet: Llama seminal plasma protein separation on SDS-PAGE after exposure to different treatments in Experiment 2 (A) and Experiment 3 (B) . Seminal plasma samples were reduced, denatured, and separated on 12% polyacrylamide gel and stained with Coomassie Blue R-250. A) Lane 1 was loaded with a molecular mass standard. The remaining lanes were loaded with 20 μg of seminal plasma (SP): Lane 2 - non-treated SP; Lane 3 - SP kept at 38°C for 1 hour; Lane 4 - SP heated to 65°C for 10 min.; Lane 5 - SP heated to 65°C for 1 hour; Lane 6 -- SP treated with charcoal dextran at 4°C for 12 hours; Lane 7 -- SP treated with proteinase K (500 μg/ml) at 38°C for 1 hour; Lane 8 -- phosphate buffered saline (PBS) plus proteinase K (500 μg/ml) at 38°C for 1 hour; * Protein band represents proteinase K. B) Lane 1 was loaded with a molecular mass standard. The remaining lanes were loaded with 30 μg of seminal plasma: Lane 2 - non-treated seminal plasma (SP); Lane 3 - SP kept at 38°C for 12 hours; Lane 4 - phosphate buffered saline (PBS) plus pronase E (500 μg/ml) at 38°C for 1 hour; Lane 5, 6, 7, 8, 9 - Seminal plasma treated with pronase E (500 μg/ml) at 38°C for 1, 3, 6, 9 or 12 hours, respectively. * Protein bands represent pronase E.

Techniques Used: SDS Page, Staining

46) Product Images from "Lipopolysaccharide induced conversion of recombinant prion protein"

Article Title: Lipopolysaccharide induced conversion of recombinant prion protein

Journal: Prion

doi: 10.4161/pri.28939

Figure 3. ( A ) SDS-PAGE gel of proteinase-K treated ShPrP isoform.Lane 1: LPS-generated ShPrP β . Lane 2: ShPrP (90–232). Lane 3: ShPrP digested with PK (PK:ShPrP 1:1000). Lane 4: Molecular weight ladder (molecular weights on the
Figure Legend Snippet: Figure 3. ( A ) SDS-PAGE gel of proteinase-K treated ShPrP isoform.Lane 1: LPS-generated ShPrP β . Lane 2: ShPrP (90–232). Lane 3: ShPrP digested with PK (PK:ShPrP 1:1000). Lane 4: Molecular weight ladder (molecular weights on the

Techniques Used: SDS Page, Generated, Molecular Weight

47) Product Images from "An extracellular Staphylococcus epidermidis polysaccharide: relation to Polysaccharide Intercellular Adhesin and its implication in phagocytosis"

Article Title: An extracellular Staphylococcus epidermidis polysaccharide: relation to Polysaccharide Intercellular Adhesin and its implication in phagocytosis

Journal: BMC Microbiology

doi: 10.1186/1471-2180-12-76

Influence of proteinase K, periodate and DspB treatments on PIA and 20-kDaPS. Immunofluorescence detection of PIA ( a , c , e , g ) and 20-kDaPS ( b , d , f , h ) on S. epidermidis 1457 grown as biofilm ( a , b ) after treatment with proteinase K ( c , d ), sodium meta -periodate ( e , f ) and DspB ( g , h ).
Figure Legend Snippet: Influence of proteinase K, periodate and DspB treatments on PIA and 20-kDaPS. Immunofluorescence detection of PIA ( a , c , e , g ) and 20-kDaPS ( b , d , f , h ) on S. epidermidis 1457 grown as biofilm ( a , b ) after treatment with proteinase K ( c , d ), sodium meta -periodate ( e , f ) and DspB ( g , h ).

Techniques Used: Immunofluorescence

48) Product Images from "Bone marrow stromal cells from multiple myeloma patients uniquely induce bortezomib resistant NF-?B activity in myeloma cells"

Article Title: Bone marrow stromal cells from multiple myeloma patients uniquely induce bortezomib resistant NF-?B activity in myeloma cells

Journal: Molecular Cancer

doi: 10.1186/1476-4598-9-176

MM BMSC-induced NF-κB activity is mediated by a secreted peptide factor .(A) EMSA of NF-κB activity in RPMI8226 cells cultured alone or with BMSCs from the indicated MM patients, either directly, or physically separated by a Transwell insert (
Figure Legend Snippet: MM BMSC-induced NF-κB activity is mediated by a secreted peptide factor .(A) EMSA of NF-κB activity in RPMI8226 cells cultured alone or with BMSCs from the indicated MM patients, either directly, or physically separated by a Transwell insert ("Transwell"). 10 ng/mL TNF-α was placed either directly in RPMI8226 culture or below the Transwell insert as a positive control for membrane permeability. The black line indicates a different exposure time for the same gel for TNF-α-treated samples in order to better visualize the very different levels of binding activity. (B) EMSA of NF-κB and Oct-1 DNA-binding activity in RPMI8226 cells treated with 1× conditioned media (CM) or the same CM concentrated 2-fold (2×) or 5-fold (5×) for 24 hours. (C) EMSA of NF-κB activity in primary MM cells treated with CM derived from BMSCs of the indicated MM patients. (D) EMSA of NF-κB and Oct-1 DNA-binding activity in RPMI8226 cells treated with CM that had been previously treated with proteinase K ("P"), or heat ("H") where indicated. RPMI8226 cells were treated with CM from Pt. 134, and 100 nM bortezomib was added for the last 4 hours of culture where indicated ("B"). NF-κB dimers are labeled as before.

Techniques Used: Activity Assay, Cell Culture, Positive Control, Permeability, Binding Assay, Derivative Assay, Labeling

49) Product Images from "Inhibition of Monocytic Interleukin-12 Production by Candida albicans via Selective Activation of ERK Mitogen-Activated Protein Kinase "

Article Title: Inhibition of Monocytic Interleukin-12 Production by Candida albicans via Selective Activation of ERK Mitogen-Activated Protein Kinase

Journal: Infection and Immunity

doi: 10.1128/IAI.72.5.2513-2520.2004

C. albicans  SIIF is glycoprotein in nature. Equal amounts of  C. albicans  supernatant fluid (25 μl of SIIF) were added to different groups of monocytes, and their IL-12 levels were determined. Group 1 represents normal monocytes, while group 2 represents monocytes stimulated with supernatant fluids and IFN-γ plus LPS. The stimulated monocytes were incubated with untreated supernatant fluids (group 3), proteinase K-treated supernatant fluids (group 4), inactive proteinase K-treated supernatant fluids (group 5), or alkali-treated supernatant fluids (group 6). Data shown are means ± standard deviations of duplicate experiments.
Figure Legend Snippet: C. albicans SIIF is glycoprotein in nature. Equal amounts of C. albicans supernatant fluid (25 μl of SIIF) were added to different groups of monocytes, and their IL-12 levels were determined. Group 1 represents normal monocytes, while group 2 represents monocytes stimulated with supernatant fluids and IFN-γ plus LPS. The stimulated monocytes were incubated with untreated supernatant fluids (group 3), proteinase K-treated supernatant fluids (group 4), inactive proteinase K-treated supernatant fluids (group 5), or alkali-treated supernatant fluids (group 6). Data shown are means ± standard deviations of duplicate experiments.

Techniques Used: Incubation

50) Product Images from "Lipopolysaccharide induced conversion of recombinant prion protein"

Article Title: Lipopolysaccharide induced conversion of recombinant prion protein

Journal: Prion

doi: 10.4161/pri.28939

Figure 3. ( A ) SDS-PAGE gel of proteinase-K treated ShPrP isoform.Lane 1: LPS-generated ShPrP β . Lane 2: ShPrP (90–232). Lane 3: ShPrP digested with PK (PK:ShPrP 1:1000). Lane 4: Molecular weight ladder (molecular weights on the
Figure Legend Snippet: Figure 3. ( A ) SDS-PAGE gel of proteinase-K treated ShPrP isoform.Lane 1: LPS-generated ShPrP β . Lane 2: ShPrP (90–232). Lane 3: ShPrP digested with PK (PK:ShPrP 1:1000). Lane 4: Molecular weight ladder (molecular weights on the

Techniques Used: SDS Page, Generated, Molecular Weight

51) Product Images from "Occurrence and Potential Diagnostic Applications of Serological Cross-Reactivities between Brucella and Other Alpha-Proteobacteria"

Article Title: Occurrence and Potential Diagnostic Applications of Serological Cross-Reactivities between Brucella and Other Alpha-Proteobacteria

Journal:

doi: 10.1128/CDLI.11.5.868-873.2004

ELISA reactivities of brucellosis (I) and control (N) sera against CYT and MA antigens from  S. meliloti  before and after treatment with proteinase K.
Figure Legend Snippet: ELISA reactivities of brucellosis (I) and control (N) sera against CYT and MA antigens from S. meliloti before and after treatment with proteinase K.

Techniques Used: Enzyme-linked Immunosorbent Assay

52) Product Images from "RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription"

Article Title: RNA-Dependent DNA Binding Activity of the Pur Factor, Potentially Involved in DNA Replication and Gene Transcription

Journal: Gene Expression

doi:

Proteinase K and RNase A sensitivity of the PUR complex. Mobility shift assays were carried out using the PUR probe and nuclear extracts from RSV-infected QEF, either untreated (lanes 1, 13), or pretreated with proteinase K (lane 3) or RNase A (lanes 6–9). Nuclear extracts were also preincubated in the same conditions in the presence of enzymatically inactive forms of proteinase K (K 0 , lane 4) and RNase A (A 0 , lane 12). The RNase inhibitor (RNasin) was added (lane 9) prior to incubation with RNase A.
Figure Legend Snippet: Proteinase K and RNase A sensitivity of the PUR complex. Mobility shift assays were carried out using the PUR probe and nuclear extracts from RSV-infected QEF, either untreated (lanes 1, 13), or pretreated with proteinase K (lane 3) or RNase A (lanes 6–9). Nuclear extracts were also preincubated in the same conditions in the presence of enzymatically inactive forms of proteinase K (K 0 , lane 4) and RNase A (A 0 , lane 12). The RNase inhibitor (RNasin) was added (lane 9) prior to incubation with RNase A.

Techniques Used: Mobility Shift, Infection, Incubation

53) Product Images from "Elongation Factor SII Contacts the 3?-End of RNA in the RNA Polymerase II Elongation Complex *"

Article Title: Elongation Factor SII Contacts the 3?-End of RNA in the RNA Polymerase II Elongation Complex *

Journal: The Journal of Biological Chemistry

doi:

Photoaffinity labeling of SII and RNA polymerase II polypeptides RNA polymerase II elongation complexes bearing 32 P-containing RNA were assembled, labeled, and irradiated as described in the text in the presence ( lanes 1 and 3–6 ) or absence ( lane 2 ) of recombinant human SII (TSK-phenyl-5PW, 1.25 μ g). Proteins were denatured and resolved on an 8% SDS-polyacrylamide gel. Lane 2 shows cross-linked polypeptides in the absence of added SII. Complexes in lanes 3 and 4 were treated with RNase A (10 μ g) and proteinase K ( PRK , 20 μ g), respectively, following UV irradiation. Complexes in lane 5 were treated as those in lane 1 but were not irradiated with UV. Complexes in lane 6 contained RNA synthesized using UTP rather than 4-thio-UTP ( 4SU ). Positions of prestained molecular mass markers (Bio-Rad) (myosin (203 kDa), β -galactosidase (118 kDa), bovine serum albumin (86 kDa), ovalbumin (51.6 kDa), carbonic anhydrase (34.1 kDa), soybean trypsin inhibitor (29 kDa), and lysozyme (19.2 kDa)) are indicated at left . Positions of individual cross-linked polypeptides and free RNA are indicated at right .
Figure Legend Snippet: Photoaffinity labeling of SII and RNA polymerase II polypeptides RNA polymerase II elongation complexes bearing 32 P-containing RNA were assembled, labeled, and irradiated as described in the text in the presence ( lanes 1 and 3–6 ) or absence ( lane 2 ) of recombinant human SII (TSK-phenyl-5PW, 1.25 μ g). Proteins were denatured and resolved on an 8% SDS-polyacrylamide gel. Lane 2 shows cross-linked polypeptides in the absence of added SII. Complexes in lanes 3 and 4 were treated with RNase A (10 μ g) and proteinase K ( PRK , 20 μ g), respectively, following UV irradiation. Complexes in lane 5 were treated as those in lane 1 but were not irradiated with UV. Complexes in lane 6 contained RNA synthesized using UTP rather than 4-thio-UTP ( 4SU ). Positions of prestained molecular mass markers (Bio-Rad) (myosin (203 kDa), β -galactosidase (118 kDa), bovine serum albumin (86 kDa), ovalbumin (51.6 kDa), carbonic anhydrase (34.1 kDa), soybean trypsin inhibitor (29 kDa), and lysozyme (19.2 kDa)) are indicated at left . Positions of individual cross-linked polypeptides and free RNA are indicated at right .

Techniques Used: Labeling, Irradiation, Recombinant, Synthesized

54) Product Images from "Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction"

Article Title: Characterization of Nef-CXCR4 Interactions Important for Apoptosis Induction

Journal: Journal of Virology

doi: 10.1128/JVI.78.20.11084-11096.2004

Analysis of CXCR4 binding. MDA-MB-468 cultures were transfected with Pc-Fusin or Pc-CCR5 and allowed to transiently express the specific receptor. (A) The cultures were then exposed to a fluorescently tagged (with Flc) ligand, either the Nef protein (4.3 nM) or M1 (4.2 nM) peptide, for 1 h at 37°C. The first supernatants, containing unbound fluorescently tagged ligand, were removed and assayed for fluorescence levels. The cultures were treated with proteinase K to strip off the cell membrane-bound ligand (fluorescently tagged), and these second supernatants were assayed for fluorescence levels. The resultant data were plotted for each condition to obtain a bound fluorescence (first supernatant)/unbound fluorescence (second supernatant) ratio. Bars 1 and 3 represent CXCR4-transfected MDA-MB-468 cells, and bars 2 and 4 represent CCR5-transfected MDA-MB-468 cells. Bars 1 and 2 represent cultures exposed to the fluorescently tagged motif 1 peptide; bars 3 and 4 represent cultures exposed to the fluorescently tagged Nef protein. (B) The cultures were exposed to tagged Nef protein (4.3 nM) alone or tagged Nef protein with one of the following competitive ligands: SDF-1α (4.7 nM), CXCR4 antibody (5 μg/ml), or CCR5 antibody (5 μg/ml). The supernatants were collected and assayed as described above. Bars 1 to 4 represent CXCR4-transfected MDA-MB-468 cells, and bars 5 to 8 represent CCR5-transfected MDA-MB-468 cells. Bars 1 and 5 represent cells exposed to tagged Nef alone, bars 2 and 6 represent cells exposed to tagged Nef and CCR5 antibody, bars 3 and 7 represent cells exposed to tagged Nef and CXCR4 antibody, and bars 4 and 8 represent cells exposed to tagged Nef and SDF-1α. The error bars show the standard errors of the measurements, and the results are a compilation of at least two independent experiments.
Figure Legend Snippet: Analysis of CXCR4 binding. MDA-MB-468 cultures were transfected with Pc-Fusin or Pc-CCR5 and allowed to transiently express the specific receptor. (A) The cultures were then exposed to a fluorescently tagged (with Flc) ligand, either the Nef protein (4.3 nM) or M1 (4.2 nM) peptide, for 1 h at 37°C. The first supernatants, containing unbound fluorescently tagged ligand, were removed and assayed for fluorescence levels. The cultures were treated with proteinase K to strip off the cell membrane-bound ligand (fluorescently tagged), and these second supernatants were assayed for fluorescence levels. The resultant data were plotted for each condition to obtain a bound fluorescence (first supernatant)/unbound fluorescence (second supernatant) ratio. Bars 1 and 3 represent CXCR4-transfected MDA-MB-468 cells, and bars 2 and 4 represent CCR5-transfected MDA-MB-468 cells. Bars 1 and 2 represent cultures exposed to the fluorescently tagged motif 1 peptide; bars 3 and 4 represent cultures exposed to the fluorescently tagged Nef protein. (B) The cultures were exposed to tagged Nef protein (4.3 nM) alone or tagged Nef protein with one of the following competitive ligands: SDF-1α (4.7 nM), CXCR4 antibody (5 μg/ml), or CCR5 antibody (5 μg/ml). The supernatants were collected and assayed as described above. Bars 1 to 4 represent CXCR4-transfected MDA-MB-468 cells, and bars 5 to 8 represent CCR5-transfected MDA-MB-468 cells. Bars 1 and 5 represent cells exposed to tagged Nef alone, bars 2 and 6 represent cells exposed to tagged Nef and CCR5 antibody, bars 3 and 7 represent cells exposed to tagged Nef and CXCR4 antibody, and bars 4 and 8 represent cells exposed to tagged Nef and SDF-1α. The error bars show the standard errors of the measurements, and the results are a compilation of at least two independent experiments.

Techniques Used: Binding Assay, Multiple Displacement Amplification, Transfection, Fluorescence, Stripping Membranes

55) Product Images from "Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids"

Article Title: Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

Journal: Scientific Reports

doi: 10.1038/srep34048

IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf 21 microsomal membranes. ( a ) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. ( b ) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. ( c–e ) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c ) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. ( d,e ) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14 C-leucine supplementation.
Figure Legend Snippet: IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf 21 microsomal membranes. ( a ) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. ( b ) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. ( c–e ) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c ) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. ( d,e ) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14 C-leucine supplementation.

Techniques Used: Fluorescence, Standard Deviation, Autoradiography, Isotopic Labeling

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TUNEL Assay:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: .. In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK). .. TUNEL was visualized using avidin-biotinylated horseradish complex (ABC, Vector Laboratories, Peterborough, UK) and diaminobenzidine/H2 O2 (DAB, Sigma, Poole, UK).

DNA Extraction:

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Article Snippet: Paragraph title: DNA isolation. ... The virus suspension was incubated overnight in 0.5% sodium dodecyl sulfate with 0.05 μl of protease K (Promega, Madison, Wis.) per ml at 65°C.

Immunohistochemistry:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: Paragraph title: Histology and immunohistochemistry ... In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK).

Concentration Assay:

Article Title: Discovery and identification of potential biomarkers of pediatric Acute Lymphoblastic Leukemia
Article Snippet: Protease K (0.1 μg, Promega Corporation, USA) was then added into the sample solution and incubated for 45 min at 37°C. .. The digestion was stopped by adding formic acid to a final concentration of 0.1%.

Biomarker Assay:

Article Title: Discovery and identification of potential biomarkers of pediatric Acute Lymphoblastic Leukemia
Article Snippet: Identification of candidate protein biomarkers by LC-MS/MS In-solution digestion of each concentrated fraction, which contains one candidate protein biomarker, was performed with a standard protocol. .. Protease K (0.1 μg, Promega Corporation, USA) was then added into the sample solution and incubated for 45 min at 37°C.

Avidin-Biotin Assay:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK). .. TUNEL was visualized using avidin-biotinylated horseradish complex (ABC, Vector Laboratories, Peterborough, UK) and diaminobenzidine/H2 O2 (DAB, Sigma, Poole, UK).

Blocking Assay:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK). .. For immunohistochemistry, sections were rehydrated, heat treatment used for antigen retrieval and following blocking with appropriate serum together with 0.1% Triton in PBS, sections were incubated with primary antibody overnight at 4°C: CCasp-3 (1:250: Cell Signalling, New England Biolabs, Herts, UK), Iba-1 (1:1000; Wako, Osaka, Japan) or GFAP (1:1000, Invitrogen, Paisley, UK).

Activation Assay:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: Glial activation was assessed by staining for microglial ionized calcium binding molecule 1 (Iba-1) and astrocyte glial fibrillary acidic protein (GFAP) immunoreactivity. .. In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK).

Incubation:

Article Title: Discovery and identification of potential biomarkers of pediatric Acute Lymphoblastic Leukemia
Article Snippet: .. Protease K (0.1 μg, Promega Corporation, USA) was then added into the sample solution and incubated for 45 min at 37°C. .. The digestion was stopped by adding formic acid to a final concentration of 0.1%.

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: .. In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK). .. TUNEL was visualized using avidin-biotinylated horseradish complex (ABC, Vector Laboratories, Peterborough, UK) and diaminobenzidine/H2 O2 (DAB, Sigma, Poole, UK).

Article Title: Detection and Heterogeneity of Herpesviruses Causing Pacheco's Disease in Parrots
Article Snippet: .. The virus suspension was incubated overnight in 0.5% sodium dodecyl sulfate with 0.05 μl of protease K (Promega, Madison, Wis.) per ml at 65°C. ..

Mass Spectrometry:

Article Title: Discovery and identification of potential biomarkers of pediatric Acute Lymphoblastic Leukemia
Article Snippet: Protease K (0.1 μg, Promega Corporation, USA) was then added into the sample solution and incubated for 45 min at 37°C. .. The LTQ mass spectrometer was operated in the data-dependent mode in which first the initial MS scan recorded the mass to charge (m/z) ratios of ions over the mass range from 400–2000 Da.

Polymerase Chain Reaction:

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Article Snippet: .. Genotyping For first part of the study (flux-pool), DNA for PCR was extracted from cells from the tip of the tail (5 mm) after overnight digestion at 56°C with protease K (200 μg/ml) (Promega, Charbonnière, France) freshly added in a buffer solution containing 100 mM Tris (pH 8.5), 200 mM NaCl, 5 mM EDTA, and 0.2% SDS. ..

Staining:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: Two sections approximate to Bregma levels 00 and -2.0 (5mm apart) were assessed for each animal for each stain. .. In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK).

Binding Assay:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: Glial activation was assessed by staining for microglial ionized calcium binding molecule 1 (Iba-1) and astrocyte glial fibrillary acidic protein (GFAP) immunoreactivity. .. In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK).

Plasmid Preparation:

Article Title: Isoflurane Exposure Induces Cell Death, Microglial Activation and Modifies the Expression of Genes Supporting Neurodevelopment and Cognitive Function in the Male Newborn Piglet Brain
Article Snippet: In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK). .. In brief, TUNEL sections were pre-treated for 15mins in 3% hydrogen peroxide, subjected to a 15mins peptidase pre-digestion with 20μg ml-1 protease K (Promega, Southampton, UK) at 65°C and incubated for 2h at 37°C with TUNEL solution (Roche, Burgess Hill, UK).

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    Promega proteinase k
    Integrin engagement and activation of the integrin/Src/paxillin signaling pathway by the gH/gL/UL128-131 complex allows efficient HCMV internalization into target monocytes. (A and B) Monocytes were pretreated with 1 µM PP2, 1 µM AG1478, 5 µg/ml of blocking anti-β1 or anti-β3 integrin antibodies, or 5 µg/ml of IgG for 1 h at 37°C/5% CO 2 . (C and D) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (A, B, C, and D) Monocytes were then HCMV (BADwt or BADrUL131)-infected (M.O.I. of 0.1) for 1 h at 4°C, washed, treated with 5 U/ml of α-thrombin (D only), then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with <t>Proteinase</t> K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p
    Proteinase K, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega granzyme cleavage
    Probing <t>granzyme</t> B cleavage efficiencies of Bid and BNIP-2 in cell-free lysates. Alkylated freeze-thaw lysates of human Jurkat and mouse YAC-1 cells were incubated with serial dilutions (ranging from 1 μM to 0.97 nM (from right to left)) of human and mouse granzyme B to assess BNIP-2 ( A : Jurkat) and Bid ( B : Jurkat and C : YAC-1) cleavage. Precursors are indicated by black arrows, whereas dashed and grey arrows represent GrB generated BNIP-2 cleavage fragments. BNIP-2 results in the YAC-1 background are lacking since no endogenous BNIP-2 precursors could be detected by means of immunoblotting (data not shown).
    Granzyme Cleavage, supplied by Promega, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integrin engagement and activation of the integrin/Src/paxillin signaling pathway by the gH/gL/UL128-131 complex allows efficient HCMV internalization into target monocytes. (A and B) Monocytes were pretreated with 1 µM PP2, 1 µM AG1478, 5 µg/ml of blocking anti-β1 or anti-β3 integrin antibodies, or 5 µg/ml of IgG for 1 h at 37°C/5% CO 2 . (C and D) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (A, B, C, and D) Monocytes were then HCMV (BADwt or BADrUL131)-infected (M.O.I. of 0.1) for 1 h at 4°C, washed, treated with 5 U/ml of α-thrombin (D only), then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Journal: PLoS Pathogens

    Article Title: The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

    doi: 10.1371/journal.ppat.1003463

    Figure Lengend Snippet: Integrin engagement and activation of the integrin/Src/paxillin signaling pathway by the gH/gL/UL128-131 complex allows efficient HCMV internalization into target monocytes. (A and B) Monocytes were pretreated with 1 µM PP2, 1 µM AG1478, 5 µg/ml of blocking anti-β1 or anti-β3 integrin antibodies, or 5 µg/ml of IgG for 1 h at 37°C/5% CO 2 . (C and D) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (A, B, C, and D) Monocytes were then HCMV (BADwt or BADrUL131)-infected (M.O.I. of 0.1) for 1 h at 4°C, washed, treated with 5 U/ml of α-thrombin (D only), then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Article Snippet: Briefly, monocytes, fibroblasts, or epithelial cells were treated and then HCMV infected (M.O.I. of 0.1) for 1 h at 4°C, washed with 1× PBS (Mediatech, Inc.) and temperature shifted to 37°C for 1 h. Cells were washed and treated with 2 mg/ml solution of Proteinase K (Promega) for 1 h at 4°C.

    Techniques: Activation Assay, Blocking Assay, Transfection, Infection, Real-time Polymerase Chain Reaction

    Presence of the gH/gL/UL128-131 complex links HCMV′s ability to activate and efficiently enter into target monocytes. (A) Approximately 2×10 6 virions of Towne (p.40, p.51 and p.57), AD169, TB40/E and TB40/F were spun down through a sucrose cushion, lysed and western blot analyses were performed using antibodies recognizing the HCMV proteins, pp65 and pUL130. (B) Monocytes were isolated and cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src and p70 S6 kinase. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (C) Monocytes were mock infected or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169) infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and semi-quantitative PCR was performed using primers complementary to genomic HCMV DNA and cellular GAPDH, as an internal control. PCR reactions were analyzed by agarose gel electrophoresis using ethidium bromide. (D, E, F, G, H, I and J) Monocytes were mock- or HCMV (TB40/E or AD169)-infected (M.O.I. of 5) for 1 h at 4°C, then 2 mM of DTSSP [3,3′-dithiobis (sulfosuccinimidylpropionate] was added at 4°C for additional 2 h. Cells were spun down and lysed. Antibodies recognizing β1, β3 integrins, HCMV gH or isotype control IgG were added overnight at 4°C to cellular lysates and then protein A/G Sepharose was added for 4 h at 4°C. Protein A/G Sepharose beads with bound protein complexes were spun down, washed with a lysis buffer and resuspended in sample buffer. Western blot analyses were performed using antibodies recognizing β1 and β3 integrins, as well as the HCMV gH and pUL130. Lysates from HCMV-infected monocytes were also analyzed for equal levels of β1, β3 integrins and actin in samples undergoing immunoprecipitation. All experiments were repeated at least three times and representative results are shown. Note: The arrows point to the band of interest. The asterisks mark non-specific bands. (K) The schematic diagram describes our cumulative data from the immunoprecipitation analysis; illustrating the interaction between the gH/gL/UL128-131 complex of TB40/E strain or gH/gL/(gO) complex of AD169 strain with cellular integrins.

    Journal: PLoS Pathogens

    Article Title: The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

    doi: 10.1371/journal.ppat.1003463

    Figure Lengend Snippet: Presence of the gH/gL/UL128-131 complex links HCMV′s ability to activate and efficiently enter into target monocytes. (A) Approximately 2×10 6 virions of Towne (p.40, p.51 and p.57), AD169, TB40/E and TB40/F were spun down through a sucrose cushion, lysed and western blot analyses were performed using antibodies recognizing the HCMV proteins, pp65 and pUL130. (B) Monocytes were isolated and cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src and p70 S6 kinase. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (C) Monocytes were mock infected or HCMV (Towne p.40, Towne p.57, TB40/E, TB40/F, AD169) infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and semi-quantitative PCR was performed using primers complementary to genomic HCMV DNA and cellular GAPDH, as an internal control. PCR reactions were analyzed by agarose gel electrophoresis using ethidium bromide. (D, E, F, G, H, I and J) Monocytes were mock- or HCMV (TB40/E or AD169)-infected (M.O.I. of 5) for 1 h at 4°C, then 2 mM of DTSSP [3,3′-dithiobis (sulfosuccinimidylpropionate] was added at 4°C for additional 2 h. Cells were spun down and lysed. Antibodies recognizing β1, β3 integrins, HCMV gH or isotype control IgG were added overnight at 4°C to cellular lysates and then protein A/G Sepharose was added for 4 h at 4°C. Protein A/G Sepharose beads with bound protein complexes were spun down, washed with a lysis buffer and resuspended in sample buffer. Western blot analyses were performed using antibodies recognizing β1 and β3 integrins, as well as the HCMV gH and pUL130. Lysates from HCMV-infected monocytes were also analyzed for equal levels of β1, β3 integrins and actin in samples undergoing immunoprecipitation. All experiments were repeated at least three times and representative results are shown. Note: The arrows point to the band of interest. The asterisks mark non-specific bands. (K) The schematic diagram describes our cumulative data from the immunoprecipitation analysis; illustrating the interaction between the gH/gL/UL128-131 complex of TB40/E strain or gH/gL/(gO) complex of AD169 strain with cellular integrins.

    Article Snippet: Briefly, monocytes, fibroblasts, or epithelial cells were treated and then HCMV infected (M.O.I. of 0.1) for 1 h at 4°C, washed with 1× PBS (Mediatech, Inc.) and temperature shifted to 37°C for 1 h. Cells were washed and treated with 2 mg/ml solution of Proteinase K (Promega) for 1 h at 4°C.

    Techniques: Western Blot, Isolation, Cell Culture, Infection, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Lysis, Immunoprecipitation

    Presence of the gH/gL/UL128-131 complex has no effect on the integrin/Src/paxillin signaling pathway in and HCMV internalization into fibroblasts. (A) Fibroblasts were cultured in low serum for 24 h at 37°C/5% CO 2 . Fibroblasts were then mock- or HCMV (BADwt or BADrUL131)-infected (M.O.I. of 5) and harvested at 20 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, and Erk. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then left at 4°C or temperature shifted to 37°C for 1 h. (C) Fibroblasts were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. (B and C) Then, fibroblasts were washed and treated with Proteinase K solution for 1 h. Cells were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Journal: PLoS Pathogens

    Article Title: The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

    doi: 10.1371/journal.ppat.1003463

    Figure Lengend Snippet: Presence of the gH/gL/UL128-131 complex has no effect on the integrin/Src/paxillin signaling pathway in and HCMV internalization into fibroblasts. (A) Fibroblasts were cultured in low serum for 24 h at 37°C/5% CO 2 . Fibroblasts were then mock- or HCMV (BADwt or BADrUL131)-infected (M.O.I. of 5) and harvested at 20 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, and Erk. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then left at 4°C or temperature shifted to 37°C for 1 h. (C) Fibroblasts were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. Fibroblasts were infected with BADwt or BADrUL131 (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. (B and C) Then, fibroblasts were washed and treated with Proteinase K solution for 1 h. Cells were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Article Snippet: Briefly, monocytes, fibroblasts, or epithelial cells were treated and then HCMV infected (M.O.I. of 0.1) for 1 h at 4°C, washed with 1× PBS (Mediatech, Inc.) and temperature shifted to 37°C for 1 h. Cells were washed and treated with 2 mg/ml solution of Proteinase K (Promega) for 1 h at 4°C.

    Techniques: Cell Culture, Infection, Western Blot, Transfection, Real-time Polymerase Chain Reaction

    Regulation of the actin cytoskeleton and dynamin is essential for efficient internalization of clinical-like HCMV isolates into monocytes, but not into fibroblasts. (A) Monocytes were pretreated with DMSO, 0.5 µM jasplakinolide, or 2.5 µM latrunculin A. (B) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (C) Monocytes were preatreated with 50 µM dynasore. (D) Monocytes were transfected with siRNA complementary to dynamin or a control siRNA for 48 h. (E and F) Fibroblasts were pretreated with DMSO, 0.5 µM jasplakinolide, 2.5 µM latrunculin A, or 50 µM dynasore. (A, B, C, D, E, and F) Cells were then infected with BADwt, BADrUL131 or TB40/E (M.O.I. of 0.1) for 1 h at 4°C washed and then temperature shifted to 37°C for 1 h. Monocytes were then treated with 0.5 µM jasplakinolide at 37°C for an additional 1 h (only B). Cells were then washed and treated with Proteinase K solution for 1 h. Cells were harvested and PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA. For qPCR data, results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Journal: PLoS Pathogens

    Article Title: The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

    doi: 10.1371/journal.ppat.1003463

    Figure Lengend Snippet: Regulation of the actin cytoskeleton and dynamin is essential for efficient internalization of clinical-like HCMV isolates into monocytes, but not into fibroblasts. (A) Monocytes were pretreated with DMSO, 0.5 µM jasplakinolide, or 2.5 µM latrunculin A. (B) Monocytes were transfected with siRNA complementary to paxillin or a control siRNA for 48 h. (C) Monocytes were preatreated with 50 µM dynasore. (D) Monocytes were transfected with siRNA complementary to dynamin or a control siRNA for 48 h. (E and F) Fibroblasts were pretreated with DMSO, 0.5 µM jasplakinolide, 2.5 µM latrunculin A, or 50 µM dynasore. (A, B, C, D, E, and F) Cells were then infected with BADwt, BADrUL131 or TB40/E (M.O.I. of 0.1) for 1 h at 4°C washed and then temperature shifted to 37°C for 1 h. Monocytes were then treated with 0.5 µM jasplakinolide at 37°C for an additional 1 h (only B). Cells were then washed and treated with Proteinase K solution for 1 h. Cells were harvested and PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA. For qPCR data, results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Article Snippet: Briefly, monocytes, fibroblasts, or epithelial cells were treated and then HCMV infected (M.O.I. of 0.1) for 1 h at 4°C, washed with 1× PBS (Mediatech, Inc.) and temperature shifted to 37°C for 1 h. Cells were washed and treated with 2 mg/ml solution of Proteinase K (Promega) for 1 h at 4°C.

    Techniques: Transfection, Infection, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    The HCMV gH/gL/UL128-131 complex is critical for activation of, and for efficient and productive viral internalization into target monocytes. (A) Monocytes were cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (BAD wt or BAD r UL131)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, Erk and SAPK/JNK. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Monocytes were HCMV (BADwt, BADrUL131, TB40/F or TB40/E)-infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Journal: PLoS Pathogens

    Article Title: The HCMV gH/gL/UL128-131 Complex Triggers the Specific Cellular Activation Required for Efficient Viral Internalization into Target Monocytes

    doi: 10.1371/journal.ppat.1003463

    Figure Lengend Snippet: The HCMV gH/gL/UL128-131 complex is critical for activation of, and for efficient and productive viral internalization into target monocytes. (A) Monocytes were cultured in low serum for 24 h at 37°C/5% CO 2 . Monocytes were then mock- or HCMV (BAD wt or BAD r UL131)-infected (M.O.I. of 5) and harvested at 15 min. pi. Western blot analyses were performed using antibodies specific for the phosphorylated and non-phosphorylated forms of Src, paxillin, Erk and SAPK/JNK. Actin was used as a loading control. The results were also measured by densitometry with relative numbers shown in the figure. (B) Monocytes were HCMV (BADwt, BADrUL131, TB40/F or TB40/E)-infected (M.O.I. of 0.1) for 1 h at 4°C, then temperature shifted to 37°C for 1 h. Monocytes were washed and treated with Proteinase K solution for 1 h. Monocytes were then harvested and quantitative real-time PCR was performed using primers complementary to genomic HCMV DNA and 18S rRNA, as an internal control. Results are plotted as a mean ±SEM. Student's T-tests were performed and p

    Article Snippet: Briefly, monocytes, fibroblasts, or epithelial cells were treated and then HCMV infected (M.O.I. of 0.1) for 1 h at 4°C, washed with 1× PBS (Mediatech, Inc.) and temperature shifted to 37°C for 1 h. Cells were washed and treated with 2 mg/ml solution of Proteinase K (Promega) for 1 h at 4°C.

    Techniques: Activation Assay, Cell Culture, Infection, Western Blot, Real-time Polymerase Chain Reaction

    Inhibition of PolB1-mediated DNA strand displacement by Sso7d. ( A ) Effect of Sso7d on strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Sso7d. The reaction mixtures were treated with proteinase K and extracted with phenol/chloroform. Samples were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–9, Sso7d was added to 0, 0.5, 2.5, 10, 25, 50 and 90 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on templates bound maximally by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 or P36/L72 (2 nM) in the presence of 90 μM Sso7d. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. ( C ) Sketches of primer templates used in experiments shown in Figure 2 D. Primers P17 and P59 are annealed to C72 starting from the same base on the minicircle as that for P36. P36(5′AT-rich) and P36(5′GC-rich), which carry 5′-AT-rich and 5′-GC-rich sequences, respectively, are annealed to different regions of C72. ( D ) Template dependence of the inhibition of strand displacement by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated primer template (2 nM) in the presence or absence of Sso7d (90 μM). Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Gels were dried and exposed to radiographic film.

    Journal: Nucleic Acids Research

    Article Title: Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    doi: 10.1093/nar/gkt588

    Figure Lengend Snippet: Inhibition of PolB1-mediated DNA strand displacement by Sso7d. ( A ) Effect of Sso7d on strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Sso7d. The reaction mixtures were treated with proteinase K and extracted with phenol/chloroform. Samples were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–9, Sso7d was added to 0, 0.5, 2.5, 10, 25, 50 and 90 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on templates bound maximally by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 or P36/L72 (2 nM) in the presence of 90 μM Sso7d. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. ( C ) Sketches of primer templates used in experiments shown in Figure 2 D. Primers P17 and P59 are annealed to C72 starting from the same base on the minicircle as that for P36. P36(5′AT-rich) and P36(5′GC-rich), which carry 5′-AT-rich and 5′-GC-rich sequences, respectively, are annealed to different regions of C72. ( D ) Template dependence of the inhibition of strand displacement by Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated primer template (2 nM) in the presence or absence of Sso7d (90 μM). Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Gels were dried and exposed to radiographic film.

    Article Snippet: When a chromatin protein was included in the reaction, a solution (4 μl) containing 3% SDS, 150 mM EDTA and 15 mg/ml proteinase K (Promega) was added to the sample.

    Techniques: Inhibition, Incubation, Electrophoresis, Sequencing

    Modulation of PolB1-mediated strand displacement by Sso7d in the presence of PCNA and RFC. ( A ) PolB1-mediated DNA strand displacement. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Sso7d were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–10, Sso7d concentrations were 0.5, 2.5, 10, 25, 50, 90 μM, respectively. ( B ) PolB1-mediated RNA strand displacement. Reactions were assembled and processed as described in (A) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

    Journal: Nucleic Acids Research

    Article Title: Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    doi: 10.1093/nar/gkt588

    Figure Lengend Snippet: Modulation of PolB1-mediated strand displacement by Sso7d in the presence of PCNA and RFC. ( A ) PolB1-mediated DNA strand displacement. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Sso7d were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–10, Sso7d concentrations were 0.5, 2.5, 10, 25, 50, 90 μM, respectively. ( B ) PolB1-mediated RNA strand displacement. Reactions were assembled and processed as described in (A) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

    Article Snippet: When a chromatin protein was included in the reaction, a solution (4 μl) containing 3% SDS, 150 mM EDTA and 15 mg/ml proteinase K (Promega) was added to the sample.

    Techniques: Electrophoresis

    Effect of Sso7d on RNA strand displacement by PolB1. ( A ) Sketches of RNA-primed templates used in experiments shown in Figure 4 B and C. The 12-nt oligoribonucleotide portion of the primer was indicated with a wavy line. ( B ) RNA strand displacement by PolB1 on P36(5′RNA)/C72 in the presence of Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Sso7d. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–8, Sso7d was added to 0, 0.5, 2.5, 10, 25 and 90 μM, respectively. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. ( C ) Sizes of the products of strand displacement by PolB1 in the presence of 90 μM Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated template (2 nM) in the presence of Sso7d (90 μM) under the standard assay conditions. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. Gels were dried and exposed to radiographic film.

    Journal: Nucleic Acids Research

    Article Title: Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    doi: 10.1093/nar/gkt588

    Figure Lengend Snippet: Effect of Sso7d on RNA strand displacement by PolB1. ( A ) Sketches of RNA-primed templates used in experiments shown in Figure 4 B and C. The 12-nt oligoribonucleotide portion of the primer was indicated with a wavy line. ( B ) RNA strand displacement by PolB1 on P36(5′RNA)/C72 in the presence of Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Sso7d. Lane 1, control; lane 2, 90 μM Sso7d; lanes 3–8, Sso7d was added to 0, 0.5, 2.5, 10, 25 and 90 μM, respectively. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. ( C ) Sizes of the products of strand displacement by PolB1 in the presence of 90 μM Sso7d. PolB1 (20 nM) was incubated for 15 min at 65°C with an indicated template (2 nM) in the presence of Sso7d (90 μM) under the standard assay conditions. Reaction products were subjected to electrophoresis in an 8% sequencing gel in 1× TBE. Gels were dried and exposed to radiographic film.

    Article Snippet: When a chromatin protein was included in the reaction, a solution (4 μl) containing 3% SDS, 150 mM EDTA and 15 mg/ml proteinase K (Promega) was added to the sample.

    Techniques: Incubation, Electrophoresis, Sequencing

    Comparison between Cren7 and Sso7d in modulating strand displacement by PolB1. ( A ) Effect of Cren7 on DNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Cren7. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 0.64 μM Cren7; lanes 3–9, Cren7 was added to 0, 0.04, 0.08, 0.12, 0.16, 0.32 and 0.64 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on P36/L72 and P36/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated with P36/L72 or P36/C72 (2 nM) in the presence of 0.64 μM Cren7 under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1× TBE. ( C ) Effect of Cren7 on RNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Cren7. Lane 1, control; lane 2, 2.5 μM Cren7; lanes 3–10, Cren7 was added to 0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1 × TBE. ( D ) Sizes of the products of strand displacement by PolB1 on P36(5′RNA)/L72 and P36(5′RNA)/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/L72 or P36(5′RNA)/C72 (2 nM) in the presence of Cren7 (2.5 μM) under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1 × TBE. ( E ) Modulation of PolB1-mediated DNA strand displacement by Cren7 in the presence of PCNA and RFC. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Cren7 were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–11, Cren7 concentrations were 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. ( F ) Modulation of PolB1-mediated RNA strand displacement by Cren7 in the presence of PCNA and RFC. Reactions were assembled and processed as described in (E) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

    Journal: Nucleic Acids Research

    Article Title: Sulfolobus chromatin proteins modulate strand displacement by DNA polymerase B1

    doi: 10.1093/nar/gkt588

    Figure Lengend Snippet: Comparison between Cren7 and Sso7d in modulating strand displacement by PolB1. ( A ) Effect of Cren7 on DNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36/C72 (2 nM) in the presence of various amounts of Cren7. Samples were treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lane 1, control; lane 2, 0.64 μM Cren7; lanes 3–9, Cren7 was added to 0, 0.04, 0.08, 0.12, 0.16, 0.32 and 0.64 μM, respectively. ( B ) Sizes of the products of strand displacement by PolB1 on P36/L72 and P36/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated with P36/L72 or P36/C72 (2 nM) in the presence of 0.64 μM Cren7 under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1× TBE. ( C ) Effect of Cren7 on RNA strand displacement by PolB1. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/C72 (2 nM) in the presence of various amounts of Cren7. Lane 1, control; lane 2, 2.5 μM Cren7; lanes 3–10, Cren7 was added to 0, 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1 × TBE. ( D ) Sizes of the products of strand displacement by PolB1 on P36(5′RNA)/L72 and P36(5′RNA)/C72 in the presence of saturating Cren7. PolB1 (20 nM) was incubated for 15 min at 65°C with P36(5′RNA)/L72 or P36(5′RNA)/C72 (2 nM) in the presence of Cren7 (2.5 μM) under the standard assay conditions. Reaction products were resolved in an 8% sequencing gel in 1 × TBE. ( E ) Modulation of PolB1-mediated DNA strand displacement by Cren7 in the presence of PCNA and RFC. PCNA (100 nM) and RFC (100 nM) were preincubated for 5 min at 70°C with P36/C72. PolB1 (5 nM) and various amounts of Cren7 were added. After 15 min at 70°C, the mixture was treated with proteinase K and extracted with phenol/chloroform. Reaction products were subjected to electrophoresis in 8% polyacrylamide gel containing 7 M urea in 1× TBE. Lanes 5–11, Cren7 concentrations were 0.04, 0.08, 0.16, 0.32, 0.64, 1.25 and 2.5 μM, respectively. ( F ) Modulation of PolB1-mediated RNA strand displacement by Cren7 in the presence of PCNA and RFC. Reactions were assembled and processed as described in (E) except that P36(5′RNA)/C72, instead of P36/C72, was used as the primer template.

    Article Snippet: When a chromatin protein was included in the reaction, a solution (4 μl) containing 3% SDS, 150 mM EDTA and 15 mg/ml proteinase K (Promega) was added to the sample.

    Techniques: Incubation, Electrophoresis, Sequencing

    IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf 21 microsomal membranes. ( a ) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. ( b ) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. ( c–e ) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c ) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. ( d,e ) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14 C-leucine supplementation.

    Journal: Scientific Reports

    Article Title: Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

    doi: 10.1038/srep34048

    Figure Lengend Snippet: IRES-mediated cell-free synthesis of N-glycosylated EGFR-eYFP and its embedment in Sf 21 microsomal membranes. ( a ) Schematic representation of standard (top) and IRES template (bottom) including regulatory elements. ( b ) Yields of total protein and eYFP fluorescence obtained using the standard (−IRES) or IRES template (+IRES) in the absence (−PG) or presence of poly G (+PG) relative to the standard reaction (−IRES, −PG). Error bars represent the standard deviation of triplicate analysis. ( c–e ) Autoradiography of IRES-mediated synthesis reactions in the presence of poly G after electrophoretic separation. c ) Deglycosylation assay of complete reaction mixtures with Endo H or PNGase F. ( d,e ) Protease protection assay of microsomal fractions with Proteinase K in the absence (−Detergent) or presence of Triton X-100 (+Detergent). Isotopic labeling was achieved by 14 C-leucine supplementation.

    Article Snippet: The protease protection assay using Proteinase K was performed in a total volume of 10 μl, containing 5 μl aliquots of the microsomal fraction resuspended in PBS, 10 ng/μl Proteinase K (Promega), 10 mM CaCl2 and either 1% Triton X-100 or PBS.

    Techniques: Fluorescence, Standard Deviation, Autoradiography, Isotopic Labeling

    Probing granzyme B cleavage efficiencies of Bid and BNIP-2 in cell-free lysates. Alkylated freeze-thaw lysates of human Jurkat and mouse YAC-1 cells were incubated with serial dilutions (ranging from 1 μM to 0.97 nM (from right to left)) of human and mouse granzyme B to assess BNIP-2 ( A : Jurkat) and Bid ( B : Jurkat and C : YAC-1) cleavage. Precursors are indicated by black arrows, whereas dashed and grey arrows represent GrB generated BNIP-2 cleavage fragments. BNIP-2 results in the YAC-1 background are lacking since no endogenous BNIP-2 precursors could be detected by means of immunoblotting (data not shown).

    Journal: BMC Biochemistry

    Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

    doi: 10.1186/1471-2091-15-21

    Figure Lengend Snippet: Probing granzyme B cleavage efficiencies of Bid and BNIP-2 in cell-free lysates. Alkylated freeze-thaw lysates of human Jurkat and mouse YAC-1 cells were incubated with serial dilutions (ranging from 1 μM to 0.97 nM (from right to left)) of human and mouse granzyme B to assess BNIP-2 ( A : Jurkat) and Bid ( B : Jurkat and C : YAC-1) cleavage. Precursors are indicated by black arrows, whereas dashed and grey arrows represent GrB generated BNIP-2 cleavage fragments. BNIP-2 results in the YAC-1 background are lacking since no endogenous BNIP-2 precursors could be detected by means of immunoblotting (data not shown).

    Article Snippet: Granzyme cleavage of in vitro transcribed and translated BNIP-2 Vectors encoding wild type BNIP-2 (with or without 5′ leader) and BNIP-2 variants were used as templates for in vitro coupled transcription/translation in a rabbit reticulocyte lysate system according to the manufacturer’s instructions (IVTT; Promega) to generate [35 S] methionine-labeled BNIP-2 (variants).

    Techniques: Incubation, Generated

    Granzyme B mediated cleavage of a peptide substrate holding the hBNIP-2 cleavage motif. Cleavage progression of the hBNIP-2 peptide substrate NH 2 .LPEDDSIEADILAITGY(NO 2 )R.OH was assayed for 2 h at 37°C with various concentrations of human and mouse GrB (50, 200 and 500 nM) at a substrate concentration of 100 μM, and its precursor and fragment peptides separated by RP-HPLC (chromatograms with absorbance at 214 nm are shown). The grey arrow indicates (residual) precursor peptide, whereas black and dashed arrows indicate peptide fragments generated upon human (upper panel) and mouse (lower panel) GrB mediated cleavage at IEAD (the dashed arrow corresponds to the N-terminal fragment NH 2 .LPEDDSIEAD.OH, whereas the black arrow corresponds to the C-terminal fragment NH 2 .ILAITGY(NO 2 )R.OH).

    Journal: BMC Biochemistry

    Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

    doi: 10.1186/1471-2091-15-21

    Figure Lengend Snippet: Granzyme B mediated cleavage of a peptide substrate holding the hBNIP-2 cleavage motif. Cleavage progression of the hBNIP-2 peptide substrate NH 2 .LPEDDSIEADILAITGY(NO 2 )R.OH was assayed for 2 h at 37°C with various concentrations of human and mouse GrB (50, 200 and 500 nM) at a substrate concentration of 100 μM, and its precursor and fragment peptides separated by RP-HPLC (chromatograms with absorbance at 214 nm are shown). The grey arrow indicates (residual) precursor peptide, whereas black and dashed arrows indicate peptide fragments generated upon human (upper panel) and mouse (lower panel) GrB mediated cleavage at IEAD (the dashed arrow corresponds to the N-terminal fragment NH 2 .LPEDDSIEAD.OH, whereas the black arrow corresponds to the C-terminal fragment NH 2 .ILAITGY(NO 2 )R.OH).

    Article Snippet: Granzyme cleavage of in vitro transcribed and translated BNIP-2 Vectors encoding wild type BNIP-2 (with or without 5′ leader) and BNIP-2 variants were used as templates for in vitro coupled transcription/translation in a rabbit reticulocyte lysate system according to the manufacturer’s instructions (IVTT; Promega) to generate [35 S] methionine-labeled BNIP-2 (variants).

    Techniques: Concentration Assay, High Performance Liquid Chromatography, Generated

    Autoradiographs showing GrB induced cleavage of murinized human BNIP-2 variants. P3′ (A) and P1′ (B) differing primed site residues following the identified P4-P1 cleavage site IEAD in hBNIP-2 were mutated to their corresponding mBNIP-2 amino acids. The in vitro transcribed and translated (mutated) BNIP-2 variants were incubated with varying concentrations (ranging from 1.95 nM to 1 μM (from left to right)) of human or mouse granzyme B. Black arrows indicate BNIP-2 precursor patterns, whereas dashed and grey arrows are indicative for BNIP-2 cleavage fragments. Percentages of cleavage are shown in the progression curves of hGrB and mGrB cleavage and indicated by full and dashed lines respectively.

    Journal: BMC Biochemistry

    Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

    doi: 10.1186/1471-2091-15-21

    Figure Lengend Snippet: Autoradiographs showing GrB induced cleavage of murinized human BNIP-2 variants. P3′ (A) and P1′ (B) differing primed site residues following the identified P4-P1 cleavage site IEAD in hBNIP-2 were mutated to their corresponding mBNIP-2 amino acids. The in vitro transcribed and translated (mutated) BNIP-2 variants were incubated with varying concentrations (ranging from 1.95 nM to 1 μM (from left to right)) of human or mouse granzyme B. Black arrows indicate BNIP-2 precursor patterns, whereas dashed and grey arrows are indicative for BNIP-2 cleavage fragments. Percentages of cleavage are shown in the progression curves of hGrB and mGrB cleavage and indicated by full and dashed lines respectively.

    Article Snippet: Granzyme cleavage of in vitro transcribed and translated BNIP-2 Vectors encoding wild type BNIP-2 (with or without 5′ leader) and BNIP-2 variants were used as templates for in vitro coupled transcription/translation in a rabbit reticulocyte lysate system according to the manufacturer’s instructions (IVTT; Promega) to generate [35 S] methionine-labeled BNIP-2 (variants).

    Techniques: In Vitro, Incubation

    Domain architecture and sequences of human and mouse BNIP-2. A . Known or putative functional motifs indicated are: the Rho-binding domain (RBD), Cdc42/Rac1-Interactive Binding (CRIB)-like motif, BCH signature motif, BCH/BCH interaction motif, caspase and granzyme cleavage sites and kinesin-targeting motifs. Annotation of the RBD is based on close similarity to known RBDs in BNIP-2 homologs and Cdc42GAP. B . ClustalW multiple sequence alignment of human and mouse BNIP-2 splice variants, i.e., the database annotated hBNIP2 sequence [Swiss-Prot: Q12982] as well as short mBNIP-2 [Trembl: Q91VL0] and long mBNIP-2 [Swiss-Prot: O54940]. The black box highlights the P4-P9′ motif with the P4-P1 IEAD GrB cleavage motif and BNIP-2 domains indicated as in A .

    Journal: BMC Biochemistry

    Article Title: Importance of extended protease substrate recognition motifs in steering BNIP-2 cleavage by human and mouse granzymes B

    doi: 10.1186/1471-2091-15-21

    Figure Lengend Snippet: Domain architecture and sequences of human and mouse BNIP-2. A . Known or putative functional motifs indicated are: the Rho-binding domain (RBD), Cdc42/Rac1-Interactive Binding (CRIB)-like motif, BCH signature motif, BCH/BCH interaction motif, caspase and granzyme cleavage sites and kinesin-targeting motifs. Annotation of the RBD is based on close similarity to known RBDs in BNIP-2 homologs and Cdc42GAP. B . ClustalW multiple sequence alignment of human and mouse BNIP-2 splice variants, i.e., the database annotated hBNIP2 sequence [Swiss-Prot: Q12982] as well as short mBNIP-2 [Trembl: Q91VL0] and long mBNIP-2 [Swiss-Prot: O54940]. The black box highlights the P4-P9′ motif with the P4-P1 IEAD GrB cleavage motif and BNIP-2 domains indicated as in A .

    Article Snippet: Granzyme cleavage of in vitro transcribed and translated BNIP-2 Vectors encoding wild type BNIP-2 (with or without 5′ leader) and BNIP-2 variants were used as templates for in vitro coupled transcription/translation in a rabbit reticulocyte lysate system according to the manufacturer’s instructions (IVTT; Promega) to generate [35 S] methionine-labeled BNIP-2 (variants).

    Techniques: Functional Assay, Binding Assay, Sequencing