proteinase k inhibitor  (Millipore)


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    Structured Review

    Millipore proteinase k inhibitor
    Detection of CDK2 in HBV capsids purified from HepG2 cells by Western blotting. HBV capsids (0.5 μg) were digested with <t>proteinase</t> K (PK) agarose beads as described in Materials and Methods. Proteinase K was then inactivated by the addition of the proteinase K inhibitor, and the sample was resolved by SDS-PAGE (lanes 8 and 10), along with the same amount of undigested capsids from the same fractions (lanes 7 and 9). Purified GST-CDK2 standards (lanes 1 to 3) and total lysate from HepG2 cells (lane 4) were loaded as controls for CDK2 detection. A sucrose fraction that did not contain HBV capsids was also treated with the proteinase K agarose beads to ensure that contaminating proteins were removed by the digestion (lane 6) or mock treated (lane 5). The capsids loaded in lanes 9 and 10 were from a capsid-positive sucrose fraction of higher purity than those loaded in lanes 7 and 8. CDK2 was detected by Western blotting using the anti-CDK2 antibody (top panel). HBc was visualized by Sypro ruby staining (bottom panel). C, core protein.
    Proteinase K Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k inhibitor/product/Millipore
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    proteinase k inhibitor - by Bioz Stars, 2020-04
    94/100 stars

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    Images

    1) Product Images from "Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids"

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

    Journal: Journal of Virology

    doi: 10.1128/JVI.01218-12

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

    Techniques Used: Purification, Western Blot, SDS Page, Staining

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

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

    Related Articles

    Centrifugation:

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids
    Article Snippet: Following the digestion, the proteinase K resin was removed by centrifugation. .. When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ).

    Agarose Gel Electrophoresis:

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids
    Article Snippet: For capsid particle analysis, proteinase K-treated capsids were loaded onto a 1% agarose gel. .. When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ).

    Labeling:

    Article Title: Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice
    Article Snippet: Proteinase-k treatment to MSC-exo Two hundred microliters of PKH26 labeled exosomes were incubated with 7 μL proteinase-K (Roche Diagnostica GmbH, Germany) and 750 μL BPS in 55 °C for 10 min [ ]. .. Proteinase-K inhibitor (7 μL) (phenylmethanesulfonyl fluoride, Sigma) was added to the solution and was suspended for 2 h in 70 ml BPS for ultracentrifugation.

    Concentration Assay:

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids
    Article Snippet: .. When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ). .. The samples were then boiled in SDS sample buffer and loaded onto a 15% SDS-PAGE gel.

    Incubation:

    Article Title: Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice
    Article Snippet: Proteinase-k treatment to MSC-exo Two hundred microliters of PKH26 labeled exosomes were incubated with 7 μL proteinase-K (Roche Diagnostica GmbH, Germany) and 750 μL BPS in 55 °C for 10 min [ ]. .. Proteinase-K inhibitor (7 μL) (phenylmethanesulfonyl fluoride, Sigma) was added to the solution and was suspended for 2 h in 70 ml BPS for ultracentrifugation.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids
    Article Snippet: The supernatant was collected and used for endogenous kinase reactions or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. .. When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ).

    Western Blot:

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids
    Article Snippet: The supernatant was collected and used for endogenous kinase reactions or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. .. When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ).

    Article Title: Intranasal administration of exosomes derived from mesenchymal stem cells ameliorates autistic-like behaviors of BTBR mice
    Article Snippet: Proteinase-K inhibitor (7 μL) (phenylmethanesulfonyl fluoride, Sigma) was added to the solution and was suspended for 2 h in 70 ml BPS for ultracentrifugation. .. Proteinase-k-treated MSC-exo were characterized with NANO-sight and Western blot.

    SDS Page:

    Article Title: Cyclin-Dependent Kinase 2 Phosphorylates S/T-P Sites in the Hepadnavirus Core Protein C-Terminal Domain and Is Incorporated into Viral Capsids
    Article Snippet: .. When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ). .. The samples were then boiled in SDS sample buffer and loaded onto a 15% SDS-PAGE gel.

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    Millipore cathepsin k inhibitor ii
    Effect of <t>cathepsin</t> K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P
    Cathepsin K Inhibitor Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 0 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cathepsin k inhibitor ii/product/Millipore
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    85
    Millipore hek cells expressing lap2 yfp or lap2 k a yfp
    Compartmentalized PRIME labeling with genetically targeted coumarin ligase. ( A ) Compartmentalized labeling of cytosolic or nuclear <t>LAP2-YFP.</t> <t>HEK</t> cells expressing both LAP2-YFP-NES and LAP2-YFP-NLS (third row), or either construct alone (first and second
    Hek Cells Expressing Lap2 Yfp Or Lap2 K A Yfp, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Price from $9.99 to $1999.99
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    94
    Millipore proteinase k inhibitor
    Detection of CDK2 in HBV capsids purified from HepG2 cells by Western blotting. HBV capsids (0.5 μg) were digested with <t>proteinase</t> K (PK) agarose beads as described in Materials and Methods. Proteinase K was then inactivated by the addition of the proteinase K inhibitor, and the sample was resolved by SDS-PAGE (lanes 8 and 10), along with the same amount of undigested capsids from the same fractions (lanes 7 and 9). Purified GST-CDK2 standards (lanes 1 to 3) and total lysate from HepG2 cells (lane 4) were loaded as controls for CDK2 detection. A sucrose fraction that did not contain HBV capsids was also treated with the proteinase K agarose beads to ensure that contaminating proteins were removed by the digestion (lane 6) or mock treated (lane 5). The capsids loaded in lanes 9 and 10 were from a capsid-positive sucrose fraction of higher purity than those loaded in lanes 7 and 8. CDK2 was detected by Western blotting using the anti-CDK2 antibody (top panel). HBc was visualized by Sypro ruby staining (bottom panel). C, core protein.
    Proteinase K Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/proteinase k inhibitor/product/Millipore
    Average 94 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    proteinase k inhibitor - by Bioz Stars, 2020-04
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    Image Search Results


    Effect of cathepsin K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Effect of cathepsin K inhibitor II on resting cell length ( A ), PS ( B ), and time to 90% relengthening ( C ) of cardiomyocytes isolated from normal diet (ND)-fed and high-fat diet (HFD)-fed wild-type mice. Data are means ± SEM, n = 50 cardiomyocytes per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Isolation, Mouse Assay

    Echocardiographic features in wild-type (WT) and cathepsin K knockout mice fed normal diet or high-fat diet. A : Left ventricular wall thickness. B : LVEDD. C : LVESD. D : Fraction shortening [(LVEDD − LVESD) / LVEDD × 100]. Mean ± SEM, n = 6–7 mice per group, * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Echocardiographic features in wild-type (WT) and cathepsin K knockout mice fed normal diet or high-fat diet. A : Left ventricular wall thickness. B : LVEDD. C : LVESD. D : Fraction shortening [(LVEDD − LVESD) / LVEDD × 100]. Mean ± SEM, n = 6–7 mice per group, * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay

    A and B : Effect of cathepsin K knockout on glucose disposal in mice fed a normal diet (ND) or a high-fat diet (HFD) after an intraperitoneal glucose (2 g/kg, body weight) challenge ( A ). Integrated area under the postglucose challenge, glucose-disposal curve ( B ). C : Effect of ND and HFD, respectively, on body weight gain over a 20-week period in wild-type and cathepsin K knockout mice. D : Representative images of wild-type (WT) and cathepsin K knockout mice fed ND or HFD and representative hearts from each group. E–H : Effect of cathepsin K knockout on HFD-induced cardiac hypertrophy. Representative images using wheat germ agglutinin staining of the left ventricular tissue ( E ), quantitation of cardiomyocyte cross-sectional area ( F ), and representative gel blots for GATA4, NFATc3, ANP, and GAPDH (loading control) ( G ), and size of isolated cardiomyocytes ( H ). Data are represented as mean ± SEM, n = 5–7 mice per group, * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: A and B : Effect of cathepsin K knockout on glucose disposal in mice fed a normal diet (ND) or a high-fat diet (HFD) after an intraperitoneal glucose (2 g/kg, body weight) challenge ( A ). Integrated area under the postglucose challenge, glucose-disposal curve ( B ). C : Effect of ND and HFD, respectively, on body weight gain over a 20-week period in wild-type and cathepsin K knockout mice. D : Representative images of wild-type (WT) and cathepsin K knockout mice fed ND or HFD and representative hearts from each group. E–H : Effect of cathepsin K knockout on HFD-induced cardiac hypertrophy. Representative images using wheat germ agglutinin staining of the left ventricular tissue ( E ), quantitation of cardiomyocyte cross-sectional area ( F ), and representative gel blots for GATA4, NFATc3, ANP, and GAPDH (loading control) ( G ), and size of isolated cardiomyocytes ( H ). Data are represented as mean ± SEM, n = 5–7 mice per group, * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay, Staining, Quantitation Assay, Aqueous Normal-phase Chromatography, Isolation

    Cardiomyocyte contractile properties in wild-type (WT) and cathepsin K knockout mice fed normal diet (ND) or high-fat diet (HFD). A and B : Representative traces from cardiomyocytes isolated from wild-type and cathepsin K knockout mice. C–H : Resting cell length, peak shortening (normalized to cell length), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt), time-to-peak shortening (TPS), and time to 90% relengthening (TR 90 ), respectively. Mean ± SEM, n = 76–87 cells per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Cardiomyocyte contractile properties in wild-type (WT) and cathepsin K knockout mice fed normal diet (ND) or high-fat diet (HFD). A and B : Representative traces from cardiomyocytes isolated from wild-type and cathepsin K knockout mice. C–H : Resting cell length, peak shortening (normalized to cell length), maximal velocity of shortening (+dL/dt), maximal velocity of relengthening (−dL/dt), time-to-peak shortening (TPS), and time to 90% relengthening (TR 90 ), respectively. Mean ± SEM, n = 76–87 cells per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Knock-Out, Mouse Assay, Isolation

    Intracellular Ca 2+ transients in cardiomyocytes and expression levels of proteins related to intracellular Ca 2+ handling in wild-type (WT) and cathepsin K knockout mice fed a normal diet (ND) or high-fat diet (HFD). A–D : Resting fura-2 fluorescence intensity (FFI), electrically stimulated increase in FFI (ΔFFI), single exponential intracellular Ca 2+ decay, and peak FFI, respectively. E : Representative Western blot images for phospho-phospholamban, phospholamban (PLB), SERCA2, and GAPDH (loading control). F–H : Densitometric quantitation of SERCA normalized to GAPDH, PLB normalized to GAPDH, and PBL-to-SERCA ratio, respectively. Mean ± SEM, n = 94–100 cells or 5–7 mice per group. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: Intracellular Ca 2+ transients in cardiomyocytes and expression levels of proteins related to intracellular Ca 2+ handling in wild-type (WT) and cathepsin K knockout mice fed a normal diet (ND) or high-fat diet (HFD). A–D : Resting fura-2 fluorescence intensity (FFI), electrically stimulated increase in FFI (ΔFFI), single exponential intracellular Ca 2+ decay, and peak FFI, respectively. E : Representative Western blot images for phospho-phospholamban, phospholamban (PLB), SERCA2, and GAPDH (loading control). F–H : Densitometric quantitation of SERCA normalized to GAPDH, PLB normalized to GAPDH, and PBL-to-SERCA ratio, respectively. Mean ± SEM, n = 94–100 cells or 5–7 mice per group. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Expressing, Knock-Out, Mouse Assay, Fluorescence, Western Blot, Quantitation Assay

    A : Immunolocalization of cathepsin K in cultured cells. H9c2 myoblasts were labeled using lysosomal dye and fluorogenic substrate MR-(LR) 2 , which detects active cathepsin K. Upper panel (merged figure) shows colocalization of cathepsin K in lysosomes under basal conditions. On stimulation with palmitic acid, active cathepsin K was released from lysosomes to the cytoplasm (indicated by arrows). B–E . Effect of cathepsin K knockout on cardiac insulin signaling molecules in normal diet (ND)-fed and high-fat diet (HFD)-fed mice. Mice were challenged with insulin (1.5 U/100 g body weight, intraperitoneally) for 10 min before isolation of the heart tissue for Western blot. Insulin-stimulated phosphorylation of Akt (indicated by an asterisk), total Akt, basal phospho-Akt, basal levels of insulin receptor-β, and glucose transporter-4 (Glut4) were assessed by Western blotting ( B ) and quantitated by densitometry ( C–E ). F and G : Effect of cathepsin K knockout on the protein levels of other cathepsins in ND-fed and HFD-fed mice. Mean ± SEM, n = 3–4 per group. WT, wild-type. * P

    Journal: Diabetes

    Article Title: Cathepsin K Knockout Mitigates High-Fat Diet-Induced Cardiac Hypertrophy and Contractile Dysfunction

    doi: 10.2337/db12-0350

    Figure Lengend Snippet: A : Immunolocalization of cathepsin K in cultured cells. H9c2 myoblasts were labeled using lysosomal dye and fluorogenic substrate MR-(LR) 2 , which detects active cathepsin K. Upper panel (merged figure) shows colocalization of cathepsin K in lysosomes under basal conditions. On stimulation with palmitic acid, active cathepsin K was released from lysosomes to the cytoplasm (indicated by arrows). B–E . Effect of cathepsin K knockout on cardiac insulin signaling molecules in normal diet (ND)-fed and high-fat diet (HFD)-fed mice. Mice were challenged with insulin (1.5 U/100 g body weight, intraperitoneally) for 10 min before isolation of the heart tissue for Western blot. Insulin-stimulated phosphorylation of Akt (indicated by an asterisk), total Akt, basal phospho-Akt, basal levels of insulin receptor-β, and glucose transporter-4 (Glut4) were assessed by Western blotting ( B ) and quantitated by densitometry ( C–E ). F and G : Effect of cathepsin K knockout on the protein levels of other cathepsins in ND-fed and HFD-fed mice. Mean ± SEM, n = 3–4 per group. WT, wild-type. * P

    Article Snippet: For experiments involving the inhibition of cathepsin K, isolated cardiomyocyte from high-fat diet–fed wild-type mice were incubated for 2 h with various concentration of cathepsin K inhibitor II (Calbiochem).

    Techniques: Cell Culture, Labeling, Knock-Out, Mouse Assay, Isolation, Western Blot

    Compartmentalized PRIME labeling with genetically targeted coumarin ligase. ( A ) Compartmentalized labeling of cytosolic or nuclear LAP2-YFP. HEK cells expressing both LAP2-YFP-NES and LAP2-YFP-NLS (third row), or either construct alone (first and second

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A fluorophore ligase for site-specific protein labeling inside living cells

    doi: 10.1073/pnas.0914067107

    Figure Lengend Snippet: Compartmentalized PRIME labeling with genetically targeted coumarin ligase. ( A ) Compartmentalized labeling of cytosolic or nuclear LAP2-YFP. HEK cells expressing both LAP2-YFP-NES and LAP2-YFP-NLS (third row), or either construct alone (first and second

    Article Snippet: HEK cells expressing LAP2-YFP (or LAP2(K → A)-YFP) were lysed under hypotonic conditions in 1 mM HEPES pH 7.5 with 5 mM MgCl2 , 1 mM phenylmethylsulphonyl fluoride, and protease inhibitor cocktail (Calbiochem).

    Techniques: Labeling, Expressing, Construct

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01218-12

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

    Article Snippet: When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ).

    Techniques: Purification, Western Blot, SDS Page, Staining

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

    Journal: Journal of Virology

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

    doi: 10.1128/JVI.01218-12

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

    Article Snippet: When proteinase K-treated capsids were resolved by SDS-PAGE, the proteinase K digestion was stopped by adding proteinase K inhibitor (Calbiochem) to 1 mM (final concentration) and incubating the sample at room temperature for 10 min ( ).

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