proteinase k Search Results


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  • 94
    Worthington Biochemical proteinase k
    Crystallography data collection and processing at FMX. Raster scans of ( a ) a bovine trypsin rod-shaped crystal and ( b ) 5–10 µm-sized <t>proteinase</t> K crystals loaded on MiTeGen loops. The raster-scan areas are shown as red-dashed rectangles; the corresponding heat maps acquired after the raster scans are shown in the insets. The grid coloring is the diffraction pattern spot count. ( c ) Clustering of the serial crystallography partial datasets based on unit-cell size. The data shown were acquired from raster scans of proteinase K microcrystals with a 200 Hz detector frame rate. Of these 279 partial datasets, 234 (blue dots) were indexed to c = 106.58 ± 0.41 Å, with 99.6% completeness (Table 1 ▸ ). The remaining 45 partial datasets (orange dots) with c = 110.06 ± 0.84 Å did not yield a complete dataset and were excluded from refinement. The unit-cell size was converted to Cartesian coordinates for better visualization. The data were clustered into two groups using a K -mean clustering algorithm. ( d ) Hierarchical cluster-analysis dendrogram for the proteinase K partial datasets acquired at a 750 Hz detector frame rate. The cutoff limit was set to 0.6 (dashed line).
    Proteinase K, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 94/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher proteinase k solution
    Initial characterization and identification of the S. caprae quorum-sensing inhibitor A . The S. caprae spent media was treated with 65 °C heat, <t>proteinase</t> K (1 mg/mL final), EGTA (5 mM final), or NaOH (to a pH of 11, then neutralized). Each sample was added to MRSA agr type I reporter to 10% final and compared to no treatment control. Florescence was measure at 24 hr, and the results are pooled from two experiments, each with N=3 replicates per condition and normalized to the media control. One-way ANOVA with multiple comparisons (Dunnett’s correction) was performed. **** indicates p
    Proteinase K Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 1330 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore proteinase k
    The factor(s) in SP responsible for TrkA activation is degraded by <t>proteinase</t> K and has a molecular weight of 30–100 kDa. (A) Seminal plasma was incubated in the presence or absence of 200 μg/ml proteinase K for 18 h at 37°C, and then spiked with PMSF to inhibit further proteolytic activity. A final concentration of 1% or 10% SP (control, mock-treated, or proteinase-K treated) was exposed for 5 min to eSF from a woman without uterine pathologies, and then probed for pTrkA pY785 levels. (B) Seminal plasma was centrifuged using filter units with nominal molecular weight limits of 3, 10, 30, and 100 kDa to generate size fractions
    Proteinase K, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 20358 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Boster Bio proteinase k
    Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml <t>proteinase</t> K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.
    Proteinase K, supplied by Boster Bio, used in various techniques. Bioz Stars score: 94/100, based on 101 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa proteinase k
    HQ formation during transcription of linear dsDNA that contained a G-core motif from the NRAS gene on the non-template strand. ( A, B ) HQ detection by DMS footprinting in DNA that carried (A) four or (B) three and two G-tracts. DNA was labeled at the 5′-end of the non-template strand with a FAM (asterisk), then subjected either to heat denaturation/renaturation (H) or transcription (T) with ATP and GTP (AG) or all four NTPs (NTP) and followed by RNase A and protease K digestion. Footprinting cleavage fragments were resolved by denaturing gel electrophoresis (left) and digitized (right) for comparison. Arrowheads indicate protected guanines. ( C ) HQ detection by native gel electrophoresis. DNA carrying four (top), three (middle) or two (bottom) G-tracts was heated or transcribed with either GTP or dzGTP and the other three NTPs as in (A, B). After RNase A and protease K digestion, the DNA was resolved on a native gel. Filled and half-filled arrowheads indicate intramolecular DNA G-quadruplexes and intermolecular DNA:RNA hybrid G-quadruplexes (HQ), respectively, and their amounts as the percentage of total DNA. ( D ) Primer extension detection of photo-cross-linking at the non-template strand by 4-thio-uridine incorporated into the RNA transcript. The 5′-FAM labeled primer was annealed to the 3′-end of the non-template of non-transcribed (N, lane 1) or transcribed (T, lane 2) DNA, followed by extension with DNA sequenase. Extension products were resolved by denaturing gel electrophoresis. G and A ladders were obtained by primer extension with ddCTP and ddTTP, respectively. Symbols on the right sides of the lanes and non-template sequence indicate the cross-linking sites (gray heart), G-tracts (black bar), guanines (filled triangle) and adenines (open triangle). Arrows on the left side of the non-template sequence indicate the sites of 4-thio-uridine incorporation. ( E ) HQ formation requires four G-tracts. Transcription and HQ detection were carried out as in (C) using dsDNA carrying the indicated G/C-tracts without or with a single mutation (nucleotide underlined) at the middle of a single G- or C-tract. Transcriptions in (A–E) were all conducted in solution containing 50 mM K +  and 40% (w/v) PEG 200.
    Proteinase K, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1159 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Abcam proteinase k
    Pathological α-synuclein is present in axonal swellings. ( A ) The effect of proteinase K treatment on the hippocampus and striatum from ASYN animals at 4 and 12 weeks post-surgery. Sections were incubated with KPBS (NT; top row) or <t>Proteinase-K</t> in KBPS for 2 hours (2 hrs; bottom row), and then immunostained with an antibody recognizing both human and rat ASYN; inserts display higher magnifications of details framed in the photos. The hippocampus showed abundant endogenous ASYN + signal in the CA3 that disappeared after 2 hours of proteinase K treatment. However, proteinase-K-resistant ASYN + aggregates (arrows) were found in striatum from animals at 4 and 12 weeks. At 4 weeks, the proteinase-K resistant formations appeared as round scattered formations in ASYN animals (arrow in inset). After 12 weeks, we observed thick fibers and rosary-like ASYN + formations, which were resistant to proteinase K digestion (arrows in inset). Scale bar = 60 µm applies to all. ( B ) P-Ser129-ASYN immunostained frontal and caudal striatal sections from ASYN animals 4 weeks (left), and 12 weeks (right) post-surgery. For each section showed in low power (top row) a high power image is presented in the bottom row to reveal details of the immunostaining in the striatum and medial forebrain bundle (MFB). In addition, each inset shows details of the P-Ser129-ASYN + structures shown in the high power photos. While at 4 weeks, single round P-Ser129-ASYN + inclusions were found in thin fibers (arrows and inset) in striatum, at 12 weeks we could find several of those round inclusions per fiber, which appeared as a rosary-like structures (arrows and inset). We also observed immunostaining in the MFB, with some single round inclusions at 4 weeks, that were smaller but more numerous at 12 weeks. Scale bar = 20 µm.
    Proteinase K, supplied by Abcam, used in various techniques. Bioz Stars score: 96/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen proteinase k
    Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) <t>Proteinase</t> K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.
    Proteinase K, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 15944 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rna grade proteinase k
    ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with <t>proteinase</t> K resistant PrP.
    Rna Grade Proteinase K, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Qiagen puregene proteinase k
    ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with <t>proteinase</t> K resistant PrP.
    Puregene Proteinase K, supplied by Qiagen, used in various techniques. Bioz Stars score: 95/100, based on 89 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Crystallography data collection and processing at FMX. Raster scans of ( a ) a bovine trypsin rod-shaped crystal and ( b ) 5–10 µm-sized proteinase K crystals loaded on MiTeGen loops. The raster-scan areas are shown as red-dashed rectangles; the corresponding heat maps acquired after the raster scans are shown in the insets. The grid coloring is the diffraction pattern spot count. ( c ) Clustering of the serial crystallography partial datasets based on unit-cell size. The data shown were acquired from raster scans of proteinase K microcrystals with a 200 Hz detector frame rate. Of these 279 partial datasets, 234 (blue dots) were indexed to c = 106.58 ± 0.41 Å, with 99.6% completeness (Table 1 ▸ ). The remaining 45 partial datasets (orange dots) with c = 110.06 ± 0.84 Å did not yield a complete dataset and were excluded from refinement. The unit-cell size was converted to Cartesian coordinates for better visualization. The data were clustered into two groups using a K -mean clustering algorithm. ( d ) Hierarchical cluster-analysis dendrogram for the proteinase K partial datasets acquired at a 750 Hz detector frame rate. The cutoff limit was set to 0.6 (dashed line).

    Journal: Journal of Synchrotron Radiation

    Article Title: High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner

    doi: 10.1107/S1600577518010354

    Figure Lengend Snippet: Crystallography data collection and processing at FMX. Raster scans of ( a ) a bovine trypsin rod-shaped crystal and ( b ) 5–10 µm-sized proteinase K crystals loaded on MiTeGen loops. The raster-scan areas are shown as red-dashed rectangles; the corresponding heat maps acquired after the raster scans are shown in the insets. The grid coloring is the diffraction pattern spot count. ( c ) Clustering of the serial crystallography partial datasets based on unit-cell size. The data shown were acquired from raster scans of proteinase K microcrystals with a 200 Hz detector frame rate. Of these 279 partial datasets, 234 (blue dots) were indexed to c = 106.58 ± 0.41 Å, with 99.6% completeness (Table 1 ▸ ). The remaining 45 partial datasets (orange dots) with c = 110.06 ± 0.84 Å did not yield a complete dataset and were excluded from refinement. The unit-cell size was converted to Cartesian coordinates for better visualization. The data were clustered into two groups using a K -mean clustering algorithm. ( d ) Hierarchical cluster-analysis dendrogram for the proteinase K partial datasets acquired at a 750 Hz detector frame rate. The cutoff limit was set to 0.6 (dashed line).

    Article Snippet: Proteinase K crystals were grown on siliconized glass cover slips (hanging drop) by equilibrating 25 mg ml−1 proteinase K (Worthington Biochemical; LS004224) in 40 mM calcium chloride, 400 mM sodium nitrate and 50 mM BisTris (pH 6.5) over a reservoir containing 160 mM calcium chloride, 1.6 M sodium nitrate and 200 mM BisTris (pH 6.5).

    Techniques:

    Initial characterization and identification of the S. caprae quorum-sensing inhibitor A . The S. caprae spent media was treated with 65 °C heat, proteinase K (1 mg/mL final), EGTA (5 mM final), or NaOH (to a pH of 11, then neutralized). Each sample was added to MRSA agr type I reporter to 10% final and compared to no treatment control. Florescence was measure at 24 hr, and the results are pooled from two experiments, each with N=3 replicates per condition and normalized to the media control. One-way ANOVA with multiple comparisons (Dunnett’s correction) was performed. **** indicates p

    Journal: Cell host & microbe

    Article Title: Coagulase-Negative Staphylococcal Strain Prevents Staphylococcus aureus Colonization and Skin Infection by Blocking Quorum Sensing

    doi: 10.1016/j.chom.2017.11.001

    Figure Lengend Snippet: Initial characterization and identification of the S. caprae quorum-sensing inhibitor A . The S. caprae spent media was treated with 65 °C heat, proteinase K (1 mg/mL final), EGTA (5 mM final), or NaOH (to a pH of 11, then neutralized). Each sample was added to MRSA agr type I reporter to 10% final and compared to no treatment control. Florescence was measure at 24 hr, and the results are pooled from two experiments, each with N=3 replicates per condition and normalized to the media control. One-way ANOVA with multiple comparisons (Dunnett’s correction) was performed. **** indicates p

    Article Snippet: For proteinase K treatment, 5 μL of a 20 mg/mL proteinase K solution (Fermentas) were added to 100 μL of spent media and incubated in a 37 °C heat block for one hour.

    Techniques:

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

    Journal: Biology of Reproduction

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

    doi: 10.1093/biolre/ioy056

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

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

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

    Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.

    Journal: Frontiers in Neuroscience

    Article Title: A PCR-Based Method for RNA Probes and Applications in Neuroscience

    doi: 10.3389/fnins.2018.00266

    Figure Lengend Snippet: Preparation and visualization of SST probe in mouse brain sections. Two-step polymerase chain reaction (PCR) amplifications were performed and PCR products were examined by agarose gel electrophoresis. (A) Products of SST from the first PCR. (B) Products of SST containing the T7 promoter from the second PCR. (C) In vitro -transcribed RNA probe of SST. (D) Staining for SST mRNA expression revealed the effect of tissue permeability on signal intensity. The left images show treatment with 1 × PBST (1% Tween-20 in 0.01 M PBS) for 20 min at room temperature (RT); the middle images show treatment with 2 μg/ml proteinase K at RT; and the right images show treatment with 2 μg/ml proteinase K at 37°C. The upper images were captured using 10 × magnification (scale bar = 500 μm), and the bottom images were captured using 20 × magnification (scale bar = 100 μm). (E) Sections were stained with different concentrations of the SST probe at 0, 0.5, 2, and 4 μg/ml, respectively. Images were captured using 10 × magnification (scale bar = 200 μm). SST, somatostatin.

    Article Snippet: The tissue sections were given a penetrating treatment with 2 μg/ml proteinase K (diluted in 1 × PBST, AR0056, Bosterbio, USA) solution for 20 min in room temperature (RT), followed by post-fixing with 4% PFA for 10 min. Then the sections were washed 3 times for 10 min each time with 1 × PBST and transferred to 2-ml RNase-free round-bottom tubes.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, In Vitro, Staining, Expressing, Permeability

    HQ formation during transcription of linear dsDNA that contained a G-core motif from the NRAS gene on the non-template strand. ( A, B ) HQ detection by DMS footprinting in DNA that carried (A) four or (B) three and two G-tracts. DNA was labeled at the 5′-end of the non-template strand with a FAM (asterisk), then subjected either to heat denaturation/renaturation (H) or transcription (T) with ATP and GTP (AG) or all four NTPs (NTP) and followed by RNase A and protease K digestion. Footprinting cleavage fragments were resolved by denaturing gel electrophoresis (left) and digitized (right) for comparison. Arrowheads indicate protected guanines. ( C ) HQ detection by native gel electrophoresis. DNA carrying four (top), three (middle) or two (bottom) G-tracts was heated or transcribed with either GTP or dzGTP and the other three NTPs as in (A, B). After RNase A and protease K digestion, the DNA was resolved on a native gel. Filled and half-filled arrowheads indicate intramolecular DNA G-quadruplexes and intermolecular DNA:RNA hybrid G-quadruplexes (HQ), respectively, and their amounts as the percentage of total DNA. ( D ) Primer extension detection of photo-cross-linking at the non-template strand by 4-thio-uridine incorporated into the RNA transcript. The 5′-FAM labeled primer was annealed to the 3′-end of the non-template of non-transcribed (N, lane 1) or transcribed (T, lane 2) DNA, followed by extension with DNA sequenase. Extension products were resolved by denaturing gel electrophoresis. G and A ladders were obtained by primer extension with ddCTP and ddTTP, respectively. Symbols on the right sides of the lanes and non-template sequence indicate the cross-linking sites (gray heart), G-tracts (black bar), guanines (filled triangle) and adenines (open triangle). Arrows on the left side of the non-template sequence indicate the sites of 4-thio-uridine incorporation. ( E ) HQ formation requires four G-tracts. Transcription and HQ detection were carried out as in (C) using dsDNA carrying the indicated G/C-tracts without or with a single mutation (nucleotide underlined) at the middle of a single G- or C-tract. Transcriptions in (A–E) were all conducted in solution containing 50 mM K +  and 40% (w/v) PEG 200.

    Journal: Nucleic Acids Research

    Article Title: Co-transcriptional formation of DNA:RNA hybrid G-quadruplex and potential function as constitutional cis element for transcription control

    doi: 10.1093/nar/gkt264

    Figure Lengend Snippet: HQ formation during transcription of linear dsDNA that contained a G-core motif from the NRAS gene on the non-template strand. ( A, B ) HQ detection by DMS footprinting in DNA that carried (A) four or (B) three and two G-tracts. DNA was labeled at the 5′-end of the non-template strand with a FAM (asterisk), then subjected either to heat denaturation/renaturation (H) or transcription (T) with ATP and GTP (AG) or all four NTPs (NTP) and followed by RNase A and protease K digestion. Footprinting cleavage fragments were resolved by denaturing gel electrophoresis (left) and digitized (right) for comparison. Arrowheads indicate protected guanines. ( C ) HQ detection by native gel electrophoresis. DNA carrying four (top), three (middle) or two (bottom) G-tracts was heated or transcribed with either GTP or dzGTP and the other three NTPs as in (A, B). After RNase A and protease K digestion, the DNA was resolved on a native gel. Filled and half-filled arrowheads indicate intramolecular DNA G-quadruplexes and intermolecular DNA:RNA hybrid G-quadruplexes (HQ), respectively, and their amounts as the percentage of total DNA. ( D ) Primer extension detection of photo-cross-linking at the non-template strand by 4-thio-uridine incorporated into the RNA transcript. The 5′-FAM labeled primer was annealed to the 3′-end of the non-template of non-transcribed (N, lane 1) or transcribed (T, lane 2) DNA, followed by extension with DNA sequenase. Extension products were resolved by denaturing gel electrophoresis. G and A ladders were obtained by primer extension with ddCTP and ddTTP, respectively. Symbols on the right sides of the lanes and non-template sequence indicate the cross-linking sites (gray heart), G-tracts (black bar), guanines (filled triangle) and adenines (open triangle). Arrows on the left side of the non-template sequence indicate the sites of 4-thio-uridine incorporation. ( E ) HQ formation requires four G-tracts. Transcription and HQ detection were carried out as in (C) using dsDNA carrying the indicated G/C-tracts without or with a single mutation (nucleotide underlined) at the middle of a single G- or C-tract. Transcriptions in (A–E) were all conducted in solution containing 50 mM K + and 40% (w/v) PEG 200.

    Article Snippet: For dimethyl sulfate (DMS) footprinting, native gel electrophoresis and photocleavage footprinting, the samples were further treated with 0.4 mg/ml of RNase A (Fermentas, MBI), then 0.6 mg/ml proteinase K (TAKARA, Dalian) at 37°C for 1 h each.

    Techniques: Footprinting, Labeling, Nucleic Acid Electrophoresis, Sequencing, Mutagenesis

    Pathological α-synuclein is present in axonal swellings. ( A ) The effect of proteinase K treatment on the hippocampus and striatum from ASYN animals at 4 and 12 weeks post-surgery. Sections were incubated with KPBS (NT; top row) or Proteinase-K in KBPS for 2 hours (2 hrs; bottom row), and then immunostained with an antibody recognizing both human and rat ASYN; inserts display higher magnifications of details framed in the photos. The hippocampus showed abundant endogenous ASYN + signal in the CA3 that disappeared after 2 hours of proteinase K treatment. However, proteinase-K-resistant ASYN + aggregates (arrows) were found in striatum from animals at 4 and 12 weeks. At 4 weeks, the proteinase-K resistant formations appeared as round scattered formations in ASYN animals (arrow in inset). After 12 weeks, we observed thick fibers and rosary-like ASYN + formations, which were resistant to proteinase K digestion (arrows in inset). Scale bar = 60 µm applies to all. ( B ) P-Ser129-ASYN immunostained frontal and caudal striatal sections from ASYN animals 4 weeks (left), and 12 weeks (right) post-surgery. For each section showed in low power (top row) a high power image is presented in the bottom row to reveal details of the immunostaining in the striatum and medial forebrain bundle (MFB). In addition, each inset shows details of the P-Ser129-ASYN + structures shown in the high power photos. While at 4 weeks, single round P-Ser129-ASYN + inclusions were found in thin fibers (arrows and inset) in striatum, at 12 weeks we could find several of those round inclusions per fiber, which appeared as a rosary-like structures (arrows and inset). We also observed immunostaining in the MFB, with some single round inclusions at 4 weeks, that were smaller but more numerous at 12 weeks. Scale bar = 20 µm.

    Journal: Scientific Reports

    Article Title: Early synaptic dysfunction induced by α-synuclein in a rat model of Parkinson’s disease

    doi: 10.1038/s41598-017-06724-9

    Figure Lengend Snippet: Pathological α-synuclein is present in axonal swellings. ( A ) The effect of proteinase K treatment on the hippocampus and striatum from ASYN animals at 4 and 12 weeks post-surgery. Sections were incubated with KPBS (NT; top row) or Proteinase-K in KBPS for 2 hours (2 hrs; bottom row), and then immunostained with an antibody recognizing both human and rat ASYN; inserts display higher magnifications of details framed in the photos. The hippocampus showed abundant endogenous ASYN + signal in the CA3 that disappeared after 2 hours of proteinase K treatment. However, proteinase-K-resistant ASYN + aggregates (arrows) were found in striatum from animals at 4 and 12 weeks. At 4 weeks, the proteinase-K resistant formations appeared as round scattered formations in ASYN animals (arrow in inset). After 12 weeks, we observed thick fibers and rosary-like ASYN + formations, which were resistant to proteinase K digestion (arrows in inset). Scale bar = 60 µm applies to all. ( B ) P-Ser129-ASYN immunostained frontal and caudal striatal sections from ASYN animals 4 weeks (left), and 12 weeks (right) post-surgery. For each section showed in low power (top row) a high power image is presented in the bottom row to reveal details of the immunostaining in the striatum and medial forebrain bundle (MFB). In addition, each inset shows details of the P-Ser129-ASYN + structures shown in the high power photos. While at 4 weeks, single round P-Ser129-ASYN + inclusions were found in thin fibers (arrows and inset) in striatum, at 12 weeks we could find several of those round inclusions per fiber, which appeared as a rosary-like structures (arrows and inset). We also observed immunostaining in the MFB, with some single round inclusions at 4 weeks, that were smaller but more numerous at 12 weeks. Scale bar = 20 µm.

    Article Snippet: As a control one slide with representative mounted section from each animal was incubated in KPBS without proteinase-K. After the proteinase-K treatment immuno-histochemical staining was performed as described above using a primary antibody raised against human and rat ASYN (sheep IgG, 1:800, Abcam) and a biotinylated secondary antibody (sheep IgG, 1:200) and developed using diaminobenzidine.

    Techniques: Incubation, Immunostaining

    ChChd3 localizes to the inner mitochondrial membrane and is required for mitochondrial fusion and crista formation. (A) Schematic diagram of Drosophila ChChd3 protein domain structure and sequence comparison of Drosophila ChChd3 with human ChChd3 and mouse ChChd3 within the CHCH domain. (B–B′′′) S2 cells showing colocalization of ChChd3 and ATP5A. Samples were stained with anti-ChChd3 and anti-ATP5A antibodies. (C) Western blot analysis of cytosolic and mitochondrial fractions separated by centrifugation. Samples were probed with anti-ChChd3, anti-ATP5A (inner membrane), and anti-tubulin antibodies. ChChd3 is enriched in the mitochondrial fraction. (D) Western blot analysis of ATP5A (detected by anti-Total OXPHOS), ChChd3, and Tom20 proteins from S2 cell mitochondria. Isolated mitochondria were treated with the indicated concentrations of digitonin followed by Proteinase K digestion. (E–F′′′) Wing imaginal discs containing control (E–E′′′) or ChChd3 D1 homozygous mutant (F–F′′′) clones stained with anti-β-Gal and anti-ATP5A antibodies. Clones are marked by the absence of β-Gal and the dashed white line. ChChd3 mutant clone cells display punctate ATP5A staining. (G) Larval body wall cells from the control larvae expressing UAS-mito-GFP with Mef2-Gal4 and showing mitochondria with tubular morphology. (H) Knockdown of ChChd3 in the larval body wall cells results in shorter mitochondria. (I and J) TEM images of mitochondria from wild-type (I) or ChChd3 D1 mutant larvae. Crista content was reduced in ChChd3 D1 mutant mitochondria. (K and L) TEM images of adult indirect flight muscle from the Mhc-Gal4 control (K) and ChChd3 knockdown flies (L). Knockdown of ChChd3 leads to fragmented mitochondria and reduced crista content. Bars, 10 μm in B; 40 μm in E; 10 μm in G; 0.1 μm in I; and 2 μm in K.

    Journal: Genetics

    Article Title: Cross-Talk Between Mitochondrial Fusion and the Hippo Pathway in Controlling Cell Proliferation During Drosophila Development

    doi: 10.1534/genetics.115.186445

    Figure Lengend Snippet: ChChd3 localizes to the inner mitochondrial membrane and is required for mitochondrial fusion and crista formation. (A) Schematic diagram of Drosophila ChChd3 protein domain structure and sequence comparison of Drosophila ChChd3 with human ChChd3 and mouse ChChd3 within the CHCH domain. (B–B′′′) S2 cells showing colocalization of ChChd3 and ATP5A. Samples were stained with anti-ChChd3 and anti-ATP5A antibodies. (C) Western blot analysis of cytosolic and mitochondrial fractions separated by centrifugation. Samples were probed with anti-ChChd3, anti-ATP5A (inner membrane), and anti-tubulin antibodies. ChChd3 is enriched in the mitochondrial fraction. (D) Western blot analysis of ATP5A (detected by anti-Total OXPHOS), ChChd3, and Tom20 proteins from S2 cell mitochondria. Isolated mitochondria were treated with the indicated concentrations of digitonin followed by Proteinase K digestion. (E–F′′′) Wing imaginal discs containing control (E–E′′′) or ChChd3 D1 homozygous mutant (F–F′′′) clones stained with anti-β-Gal and anti-ATP5A antibodies. Clones are marked by the absence of β-Gal and the dashed white line. ChChd3 mutant clone cells display punctate ATP5A staining. (G) Larval body wall cells from the control larvae expressing UAS-mito-GFP with Mef2-Gal4 and showing mitochondria with tubular morphology. (H) Knockdown of ChChd3 in the larval body wall cells results in shorter mitochondria. (I and J) TEM images of mitochondria from wild-type (I) or ChChd3 D1 mutant larvae. Crista content was reduced in ChChd3 D1 mutant mitochondria. (K and L) TEM images of adult indirect flight muscle from the Mhc-Gal4 control (K) and ChChd3 knockdown flies (L). Knockdown of ChChd3 leads to fragmented mitochondria and reduced crista content. Bars, 10 μm in B; 40 μm in E; 10 μm in G; 0.1 μm in I; and 2 μm in K.

    Article Snippet: The samples were then subjected to proteolysis with Proteinase K. Proteins were then precipitated with 10% trichloroacetic acid (TCA) and analyzed by Western blotting using rabbit anti-ChChd3 (1:2000), mouse anti-total OXPHOS (1:4000; Abcam, ab110413) and rabbit anti-Tom20 (1:1000; Proteintech, 11802-1-AP) antibodies.

    Techniques: Sequencing, Staining, Western Blot, Centrifugation, Isolation, Mutagenesis, Clone Assay, Expressing, Transmission Electron Microscopy

    Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Journal: Journal of molecular and cellular cardiology

    Article Title: HDAC1 localizes to the mitochondria of cardiac myocytes and contributes to early cardiac reperfusion injury

    doi: 10.1016/j.yjmcc.2017.12.004

    Figure Lengend Snippet: Characterization of HDAC localization in tissues and cells. (A) Class I HDAC activity in whole heart lysates and mitochondrial isolates. Acetylated substrate selective for only class I and class IIb HDACs. HDAC6 activity inhibited with 1 μM Tubastatin A (Tub A). Class I HDACs inhibited with 5 μM MS275. Activity is assessed from a 2 h. reaction. (B) Western blotting for class I HDACs in whole heart nuclear, cytoplasmic, and mitochondrial isolates. (C) Proteinase K digestion of whole heart mitochondrial isolates. (D) Western blotting for HDAC1 in skeletal muscle and liver mitochondrial isolates. (E) Staining for HDAC1 and ACAA2 in cardiac myocytes, fibroblasts and endothelial cells. Bar = 10 μm N = 3 per group for all experiments.

    Article Snippet: Mitochondria were then incubated in the presence of 50 μg/mL proteinase K (Qiagen, Germantown, MD) for 0, 20, 40 or 60 min at 37 °C.

    Techniques: Activity Assay, Western Blot, Staining

    ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with proteinase K resistant PrP.

    Journal: Prion

    Article Title: Prion Pathogenesis is Independent of Caspase-12

    doi:

    Figure Lengend Snippet: ER stress is activated in prion disease. Upregulation of ER stress markers was determined in prion infected CD1 mice (n = 2 per timepoint) after inoculation with 6.5logLD 50 RML prions. Western blot analysis was performed to analyze the levels of C12, Grp58, JNK phosphorylation, ERK phosphorylation, total PrP and proteinase K-resistant PrP. The total level of JNK, ERK, and actin were measured as loading controls. Faint processing of C12 is visible at 4 months post inoculation (mpi) but more clearly at 4.5 and 5 mpi (active fragments of C12 are indicated by an arrow head). Phosphorylation of JNK (P-JNK) as well as induction of Grp58 was observed at 4.5 and 5 mpi. Phosphorylation of ERK was observed at 4.5 and 5 mpi. Higher order SDS-resistant PrP species were first visible at 3 mpi and thereafter (an arrow head marks the migration of monomeric PrP) along with proteinase K resistant PrP.

    Article Snippet: For proteinase K treatment, 10% brain homogenates from terminally ill WT or Caspase-12 KOs were diluted to 1% in lysis buffer and treated with 50 µg/ml proteinase K (Invitrogen) for one hour at 37°C.

    Techniques: Infection, Mouse Assay, Western Blot, Migration

    Analysis of spongiform changes in C12 WT and KO brain sections (A, i and iii) show a similar amount of vacuolation, indicated by arrows, in the hippocampus of prion inoculated mice but no vacuolation in uninoculated mice [C12 WT is shown in (A, i)]. The amount of gliosis was examined by staining for GFAP, which did not show staining in uninoculated samples [C12 WT is shown in (A, iv)] but showed abundant staining in prion inoculated samples from C12 WT and C12 KO (v and vi). For all of these parameters, blinded analysis did not reveal any differences between prion inoculated C12 KO and control brains. (B) The amount of proteinase K resistant PrP was assayed in whole brain homogenates taken from prion inoculated C12 WT (n = 5) and C12 KO (n = 5) mice (treated for 50ug/ml PK for one hour at 37C), which all showed ample PK resistant PrP by immunoblotting with SAF83. Total PrP inputs are shown to ensure equivalent loading.

    Journal: Prion

    Article Title: Prion Pathogenesis is Independent of Caspase-12

    doi:

    Figure Lengend Snippet: Analysis of spongiform changes in C12 WT and KO brain sections (A, i and iii) show a similar amount of vacuolation, indicated by arrows, in the hippocampus of prion inoculated mice but no vacuolation in uninoculated mice [C12 WT is shown in (A, i)]. The amount of gliosis was examined by staining for GFAP, which did not show staining in uninoculated samples [C12 WT is shown in (A, iv)] but showed abundant staining in prion inoculated samples from C12 WT and C12 KO (v and vi). For all of these parameters, blinded analysis did not reveal any differences between prion inoculated C12 KO and control brains. (B) The amount of proteinase K resistant PrP was assayed in whole brain homogenates taken from prion inoculated C12 WT (n = 5) and C12 KO (n = 5) mice (treated for 50ug/ml PK for one hour at 37C), which all showed ample PK resistant PrP by immunoblotting with SAF83. Total PrP inputs are shown to ensure equivalent loading.

    Article Snippet: For proteinase K treatment, 10% brain homogenates from terminally ill WT or Caspase-12 KOs were diluted to 1% in lysis buffer and treated with 50 µg/ml proteinase K (Invitrogen) for one hour at 37°C.

    Techniques: Mouse Assay, Staining