hrv 3c protease Sino Biological Search Results


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    Sino Biological hrv 3c protease
    Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with <t>HRV</t> <t>3C</t> protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.
    Hrv 3c Protease, supplied by Sino Biological, used in various techniques. Bioz Stars score: 91/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hrv 3c protease/product/Sino Biological
    Average 91 stars, based on 38 article reviews
    Price from $9.99 to $1999.99
    hrv 3c protease - by Bioz Stars, 2020-08
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    92
    Sino Biological recombinant hrv 3c protease
    Interruption of RIPK1 death signaling in <t>HRV-A16</t> infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and <t>3Cpro</t> from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.
    Recombinant Hrv 3c Protease, supplied by Sino Biological, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/recombinant hrv 3c protease/product/Sino Biological
    Average 92 stars, based on 11 article reviews
    Price from $9.99 to $1999.99
    recombinant hrv 3c protease - by Bioz Stars, 2020-08
    92/100 stars
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    Image Search Results


    Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with HRV 3C protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.

    Journal: PLoS ONE

    Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

    doi: 10.1371/journal.pone.0122293

    Figure Lengend Snippet: Purification of STRA6 from Pichia pastoris . (A) Fed-batch fermentation of recombinant Pichia pastoris expressing STRA6-GFP. Changes in the wet cell weight of yeast (red line) and dissolved oxygen (blue line) are shown over time. (B) Elution profile of Ni-NTA-purified STRA6-GFP on a Superdex 200, 10/300 GL column (blue trace) and after removal of GFP by treatment with HRV 3C protease (red trace). The disappearance of the peak corresponding to STRA6-GFP and appearance of a peak corresponding to GFP are clearly visible. The concentration of protein was determined by comparing the fluorescence of the eluted fractions to a standard curve of GFP and correcting for the molecular weight of the fusion protein. The void volume was determined using the elution profiles of blue dextran. (C) A silver-stained 10% SDS gel showing the purification of STRA6-GFP. The lanes are: lane 1 , MW markers; lane 2 , microsomes, 10 μg; lane 3 , solubilized microsomes after extraction with C12E9, 10 μg; lane 4 , flow-through from Ni-NTA column, 10 μg; lane 5 , 250 mM imidazole eluate from Ni-NTA column, 3 μg; lane 6 , eluate from Superdex 200, 10/300 GL column, 3 μg. (D) Immunoblot of STRA6-GFP after purification by Ni-NTA and size exclusion chromatography (lane 1) and following treatment with HRV 3C protease to remove the GFP (lane 2) using an antibody against full-length STRA6. The positions corresponding to the predicted molecular weights of STRA6-GFP and STRA6 are indicated.

    Article Snippet: HRV 3C protease was from Sino Biological.

    Techniques: Purification, Recombinant, Expressing, Concentration Assay, Fluorescence, Molecular Weight, Staining, SDS-Gel, Flow Cytometry, Size-exclusion Chromatography

    Functional expression of STRA6-GFP in Pichia pastoris . (A) Schematic of the STRA6-GFP-c-Myc-His 6 construct designed in this study. The location of a HRV 3C protease cleavage site is indicated by the red cross. (B) Co-localization of STRA6-GFP and DyLight594-conjugated holo-RBP at the cell surface of Pichia pastoris as determined by confocal microscopy. Top panel: GFP fluorescence, indicating the location of STRA6-GFP at the cell surface. Second panel: DyLight-594 fluorescence, showing the binding of RBP to the surface of cells transformed with STRA6-GFP but not empty vector. Third panel: merged image showing the colocalization of STRA6-GFP and DyLight-594 RBP at the cell surface. Bottom panel: Differential interference contrast (DIC) image of the yeast cells. Scale bar: 5 μm (C) Co-purification of holo-RBP with broken cells isolated from yeast transformed with STRA6-GFP but not with broken cells from empty-vector-transformed cells.

    Journal: PLoS ONE

    Article Title: Production of Functional Human Vitamin A Transporter/RBP Receptor (STRA6) for Structure Determination

    doi: 10.1371/journal.pone.0122293

    Figure Lengend Snippet: Functional expression of STRA6-GFP in Pichia pastoris . (A) Schematic of the STRA6-GFP-c-Myc-His 6 construct designed in this study. The location of a HRV 3C protease cleavage site is indicated by the red cross. (B) Co-localization of STRA6-GFP and DyLight594-conjugated holo-RBP at the cell surface of Pichia pastoris as determined by confocal microscopy. Top panel: GFP fluorescence, indicating the location of STRA6-GFP at the cell surface. Second panel: DyLight-594 fluorescence, showing the binding of RBP to the surface of cells transformed with STRA6-GFP but not empty vector. Third panel: merged image showing the colocalization of STRA6-GFP and DyLight-594 RBP at the cell surface. Bottom panel: Differential interference contrast (DIC) image of the yeast cells. Scale bar: 5 μm (C) Co-purification of holo-RBP with broken cells isolated from yeast transformed with STRA6-GFP but not with broken cells from empty-vector-transformed cells.

    Article Snippet: HRV 3C protease was from Sino Biological.

    Techniques: Functional Assay, Expressing, Construct, Confocal Microscopy, Fluorescence, Binding Assay, Transformation Assay, Plasmid Preparation, Copurification, Isolation

    Interruption of RIPK1 death signaling in HRV-A16 infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: Interruption of RIPK1 death signaling in HRV-A16 infection. a Disruption of RIPK1-TRIF complexes shown by anti-TRIF immuno-precipitations, and western blots against TRIF, RIPK1 (amino acids 190), and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h). Poly I:C (5 µg/ml)-transfected control cells did not show TRIF-RIPK1 complex disruption. HC denotes heavy chain, arrow highlights co-precipitated full length RIPK1 from poly I:C-treated cells. b Time-resolved degradation of RIPK3 but not BID. Analyses by western blots against RIPK3, BID and beta-tubulin (b-Tub) from total lysates of HeLa cells infected with HRV-A16 (MOI 1) or transfected with poly I:C (5 μg/ml). c Time-resolved degradation of cIAP1 in HeLa cells infected with HRV-A16 (MOI 1) analyzed by western blots against cIAP1 and GAPDH. d Western blots against p62/SQSTM1 and beta-tubulin from lysates of untreated HeLa cells, or transfected with poly I:C (5 μg/ml), or infected with HRV-A16 (MOI 1, 24 h) ± parallel treatment with TNFα (1 μg/ml), calpain/cathepsin inhibitor E64d (10 μ m ), necrostatin-1 (Nec1, 1 μ m ), proteasomal inhibitor MG132 (10 μ m ) or AG7088 (rupintrivir, 20 n m ). p62/SQSTM1 expression is highlighted by black arrow. e Schematic depiction of dsRNA-induced death signaling (left) and HRV-A16 interception of death signaling (right). TLR3 senses dsRNA, which leads to formation of TRIF-RIPK1-FADD-p62/SQSTM1 complexes, recruitment, and proteolytic activation of pro-caspase-8 to active p30 caspase-8 (cleaved Casp8) and apoptosis (poly I:C pathway, left). This may involve binding of p30-Casp8 to RIPK1, and cleavage of an N-terminal p35 RIPK1 fragment, and p62/SQSTM1 cleavage. HRV-A16 interferes with this death signaling pathway by 3ABC and 3Cpro binding to RIPK1, and RIPK1 cleavage to an N-terminal 60 kDa fragment. RIPK1 binds to procaspase-8, dissociates from TRIF, FADD, and p62/SQSTM1 and interrupts apoptotic signaling. In addition, degradation of RIPK3, and processing of p62/SQSTM1 (distinct from poly I:C triggered p62/SQSTM1 processing) further attenuate necroptotic and NF-KB signaling. This results in virus controlled necrosis.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, Western Blot, Transfection, Expressing, Activation Assay, Binding Assay

    3Cpro generates a 60 kDa N-terminal RIPK1 fragment, and supports HRV-A16 titer production late in infection. a Western blots against RIPK1 (leucine 190), VP2, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) and treated with pan-caspase inhibitor QVD (5 µ m , 15 h), calpain/cathepsin inhibitor E64d (10 µ m , 15 h), necrostatin-1 (Nec1, 1 µ m , 15 h), AG7088 (rupintrivir, 20 n m , 15 h) or drug-combinations. The 60 kDa RIPK1 fragment is highlighted by black arrow. b In vitro cleavage of RIPK1 by 3Cpro. HeLa cell extracts were incubated with recombinant 3C protease (r3C) with or without the 3C inhibitor AG7088 (20 n m ), and analyzed by western blotting against RIPK1 (leucine 190) and lamin A/C. Reference samples included lysates from HeLa infected with HRV-A16 (MOI 1, 15 h). RIPK1 cleavage is highlighted by black arrows. c GST-RIPK1 cleavage in vitro. Recombinant GST-RIPK1 was incubated with r3C in presence or absence of AG7088 (20 n m ) or QVD (5 µ m ), and reaction products analyzed by western blotting against N-terminal RIPK1 (leucine 190), middle domain RIPK1 (amino acids 133–422), and GST. d Late addition of AG7088 inhibits production of HRV-A16. HeLa cells were infected with HRV-A16 at three different MOI (log −1, −2, −3) and treated with AG7088 at different times pi. The titer of viral progeny was determined from cells and supernatants 24 h pi for each time point by end point titration assays. Data points represent mean values from three different experiments, including SD.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: 3Cpro generates a 60 kDa N-terminal RIPK1 fragment, and supports HRV-A16 titer production late in infection. a Western blots against RIPK1 (leucine 190), VP2, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) and treated with pan-caspase inhibitor QVD (5 µ m , 15 h), calpain/cathepsin inhibitor E64d (10 µ m , 15 h), necrostatin-1 (Nec1, 1 µ m , 15 h), AG7088 (rupintrivir, 20 n m , 15 h) or drug-combinations. The 60 kDa RIPK1 fragment is highlighted by black arrow. b In vitro cleavage of RIPK1 by 3Cpro. HeLa cell extracts were incubated with recombinant 3C protease (r3C) with or without the 3C inhibitor AG7088 (20 n m ), and analyzed by western blotting against RIPK1 (leucine 190) and lamin A/C. Reference samples included lysates from HeLa infected with HRV-A16 (MOI 1, 15 h). RIPK1 cleavage is highlighted by black arrows. c GST-RIPK1 cleavage in vitro. Recombinant GST-RIPK1 was incubated with r3C in presence or absence of AG7088 (20 n m ) or QVD (5 µ m ), and reaction products analyzed by western blotting against N-terminal RIPK1 (leucine 190), middle domain RIPK1 (amino acids 133–422), and GST. d Late addition of AG7088 inhibits production of HRV-A16. HeLa cells were infected with HRV-A16 at three different MOI (log −1, −2, −3) and treated with AG7088 at different times pi. The titer of viral progeny was determined from cells and supernatants 24 h pi for each time point by end point titration assays. Data points represent mean values from three different experiments, including SD.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, Western Blot, In Vitro, Incubation, Recombinant, Titration

    HRV-A16 infection disrupts an RHIM domain-containing RIPK1-cCasp8-p62/SQSTM1 signaling complex, and leads to complex formation of 60 kDa RIPK1-Caspase-8 and 3ABC/3Cpro. a Comparison of the RHIM domain from human RIPK1, RIPK3, 3Cpro from HRV-A16/B14 by sequence alignment. Yellow indicates amino acid identities, gray similarities, * denotes conserved amino acids in the RHIM domains of RIPK1/3. b Pro-caspase-8 immuno-precipitations, and western blots against Pro-caspase-8, RIPK1 (amino acids 190) and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), or poly I:C (5 µg/ml)-treated cells, and uninfected samples. Input lysates and immuno-precipitations are shown. LC denotes light chain. c RIPK1 immuno-precipitations (leucine 190) and western blot against 3Cpro from HRV-A16-infected HeLa cells (MOI 1, 15 h) or uninfected cells. HC denotes heavy chain. d RIPK1 immuno-precipitations (leucine 190) and western blots against RIPK1, cCasp8, and p62/SQSTM1 from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) or transfected with poly I:C (5 µg/ml). Input lysates and immuno-precipitations are shown. HC denotes heavy chain, IP immuno-precipitation. Arrow highlights HRV-A16-specific p62/SQSTM1 processing.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: HRV-A16 infection disrupts an RHIM domain-containing RIPK1-cCasp8-p62/SQSTM1 signaling complex, and leads to complex formation of 60 kDa RIPK1-Caspase-8 and 3ABC/3Cpro. a Comparison of the RHIM domain from human RIPK1, RIPK3, 3Cpro from HRV-A16/B14 by sequence alignment. Yellow indicates amino acid identities, gray similarities, * denotes conserved amino acids in the RHIM domains of RIPK1/3. b Pro-caspase-8 immuno-precipitations, and western blots against Pro-caspase-8, RIPK1 (amino acids 190) and 3Cpro from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), or poly I:C (5 µg/ml)-treated cells, and uninfected samples. Input lysates and immuno-precipitations are shown. LC denotes light chain. c RIPK1 immuno-precipitations (leucine 190) and western blot against 3Cpro from HRV-A16-infected HeLa cells (MOI 1, 15 h) or uninfected cells. HC denotes heavy chain. d RIPK1 immuno-precipitations (leucine 190) and western blots against RIPK1, cCasp8, and p62/SQSTM1 from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h) or transfected with poly I:C (5 µg/ml). Input lysates and immuno-precipitations are shown. HC denotes heavy chain, IP immuno-precipitation. Arrow highlights HRV-A16-specific p62/SQSTM1 processing.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, Sequencing, Western Blot, Transfection, Immunoprecipitation

    Necrotic cell death rather than apoptosis in HRV-A16-infected primary human nasal epithelial cells. a Dose-dependent infection of hAECN with various A- and B-type rhinoviruses (HRV-A16, HRV-B14, HRV-B37, HRV-A2, HRV-A1A), and CVB3 and correlation with cell number. Values are means ± SD, n = 3. b Electron micrographs of hAECN infected with HRV-A16 (MOI 1) or treated with poly I:C or puromycin for 15 h, or uninfected cells. Proximity of vesicular structures with the cell surface indicated by black arrowheads, and swollen mitochondrial cristae in infected cells highlighted by arrows. c ROS production of HRV-A16 or HRV-A1A (MOI 1) infected cells 24 h pi, compared to luprox- (100 μ m , 1 h) or menadione (100 μ m , 1 h)-treated cells. d FACS analysis of annexin V stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. e Western blots against lamin A/C and beta-tubulin from lysates of hAECN cells infected with HRV-A16 (MOI 1, 15 h) or treated with puromycin (5 µg/ml, 15 h) with or without pan-caspase inhibitor QVD (5 µ m ). M r denotes relative molecular weight in kDa. f FACS analysis propidium iodide (PI) stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. g Western blot analysis of caspase 3, 7, 8, 9 activations in hAECN after infection with HRV-A16 (MOI 1, 15 h), poly I:C transfection (5 μg/ml) or external poly I:C addition (ext, 50 μg/ml). Arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. Infection is indicated by blotting against 3Cpro using beta-tubulin as a loading control.

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: Necrotic cell death rather than apoptosis in HRV-A16-infected primary human nasal epithelial cells. a Dose-dependent infection of hAECN with various A- and B-type rhinoviruses (HRV-A16, HRV-B14, HRV-B37, HRV-A2, HRV-A1A), and CVB3 and correlation with cell number. Values are means ± SD, n = 3. b Electron micrographs of hAECN infected with HRV-A16 (MOI 1) or treated with poly I:C or puromycin for 15 h, or uninfected cells. Proximity of vesicular structures with the cell surface indicated by black arrowheads, and swollen mitochondrial cristae in infected cells highlighted by arrows. c ROS production of HRV-A16 or HRV-A1A (MOI 1) infected cells 24 h pi, compared to luprox- (100 μ m , 1 h) or menadione (100 μ m , 1 h)-treated cells. d FACS analysis of annexin V stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. e Western blots against lamin A/C and beta-tubulin from lysates of hAECN cells infected with HRV-A16 (MOI 1, 15 h) or treated with puromycin (5 µg/ml, 15 h) with or without pan-caspase inhibitor QVD (5 µ m ). M r denotes relative molecular weight in kDa. f FACS analysis propidium iodide (PI) stainings of hAECN infected with HRV-A16 or HRV-A1A (MOI 1) for 4, 8, 15 or 24 h and comparison with poly I:C-treated cells. Values are means from approximately 10,000 cells analyzed ± SD, n = 3. g Western blot analysis of caspase 3, 7, 8, 9 activations in hAECN after infection with HRV-A16 (MOI 1, 15 h), poly I:C transfection (5 μg/ml) or external poly I:C addition (ext, 50 μg/ml). Arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. Infection is indicated by blotting against 3Cpro using beta-tubulin as a loading control.

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Infection, FACS, Western Blot, Molecular Weight, Transfection

    TLR3 is required for poly I:C-induced caspase-8 activation, which can be suppressed by HRV-A16 infection, while MDA5, TRIF, and FADD are dispensable for caspase-8 activation. a Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, 3Cpro, MDA5, and GAPDH from lysates of HeLa cells treated with siRNA against MDA5 and infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-MDA5 treatment ± pan-caspase inhibitor QVD. b Western blots against cleaved caspase-8 (cCasp8), 3Cpro, caspase-7 (Casp7 + 3Cpro serial antibody incubation), TRIF, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples and comparison of protein expression after siRNA-TRIF treatment ± pan-caspase inhibitor QVD; black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. c Western blots against cleaved caspase-8 (cCasp8), caspase-7 (Casp7), RIPK1 (aa190), FADD, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-FADD and siRNA-FADD/RIPK1 treatment. Black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. d Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, TLR3, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 µg/ml) or untreated, and comparison of protein expression after siRNA-TLR3 treatment with or without QVD (5 µ m ). Stars indicate unspecific background staining. TLR3-CT denotes C-terminal cleaved form of TLR3. e Western blots of cleaved caspase-8 (cCasp8), VP2, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-RIPK1, siRNA-TLR3 or siRNA-RIPK1/TLR3 treatment. f Knock-down of TLR3 by RNA interference in HeLa cells, analyzed by single section confocal fluorescence microscopy. Control transfections with all star siRNA are shown on the left. TLR3 immune-staining in green, nuclei (DAPI) in blue. Scale bar 30 µm. g HRV-A16 (MOI 1, 15 h)-mediated suppression of caspase-8 activation indicated by western blots against cleaved caspase-8 (cCasp8, arrow), cleaved caspase-3 (cCasp3, p17/p20, arrow), VP2, 3C, and GAPDH from lysates of HeLa cells after late addition of poly I:C (5 µg/ml, 6 h).

    Journal: Cell Death & Disease

    Article Title: Rhinovirus 3C protease suppresses apoptosis and triggers caspase-independent cell death

    doi: 10.1038/s41419-018-0306-6

    Figure Lengend Snippet: TLR3 is required for poly I:C-induced caspase-8 activation, which can be suppressed by HRV-A16 infection, while MDA5, TRIF, and FADD are dispensable for caspase-8 activation. a Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, 3Cpro, MDA5, and GAPDH from lysates of HeLa cells treated with siRNA against MDA5 and infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-MDA5 treatment ± pan-caspase inhibitor QVD. b Western blots against cleaved caspase-8 (cCasp8), 3Cpro, caspase-7 (Casp7 + 3Cpro serial antibody incubation), TRIF, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples and comparison of protein expression after siRNA-TRIF treatment ± pan-caspase inhibitor QVD; black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. c Western blots against cleaved caspase-8 (cCasp8), caspase-7 (Casp7), RIPK1 (aa190), FADD, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-FADD and siRNA-FADD/RIPK1 treatment. Black arrow highlights virus-induced caspase-7 processing product; undefined antibody background is indicated by a star. d Western blots against cleaved caspase-8 (cCasp8), cleaved caspase-3 (cCasp3), VP2, TLR3, and GAPDH from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 µg/ml) or untreated, and comparison of protein expression after siRNA-TLR3 treatment with or without QVD (5 µ m ). Stars indicate unspecific background staining. TLR3-CT denotes C-terminal cleaved form of TLR3. e Western blots of cleaved caspase-8 (cCasp8), VP2, and beta-tubulin from lysates of HeLa cells infected with HRV-A16 (MOI 1, 15 h), transfected with poly I:C (5 μg/ml) or untreated samples, and comparison of protein expression after siRNA-RIPK1, siRNA-TLR3 or siRNA-RIPK1/TLR3 treatment. f Knock-down of TLR3 by RNA interference in HeLa cells, analyzed by single section confocal fluorescence microscopy. Control transfections with all star siRNA are shown on the left. TLR3 immune-staining in green, nuclei (DAPI) in blue. Scale bar 30 µm. g HRV-A16 (MOI 1, 15 h)-mediated suppression of caspase-8 activation indicated by western blots against cleaved caspase-8 (cCasp8, arrow), cleaved caspase-3 (cCasp3, p17/p20, arrow), VP2, 3C, and GAPDH from lysates of HeLa cells after late addition of poly I:C (5 µg/ml, 6 h).

    Article Snippet: Lysates equivalent to 1.0×106 cells were incubated with four units of recombinant HRV 3C protease (Sino Biological Inc) at 4 °C for 16 h in presence or absence of inhibitors.

    Techniques: Activation Assay, Infection, Western Blot, Transfection, Expressing, Incubation, Staining, Fluorescence, Microscopy

    SDS-PAGE analysis of the CD147 antigen prior to rabbit immunization. Eukaryotic-expressed recombinant protein CD147-Fc was treated with HRV 3C protease for 16 h, and the mixture was purified by Ni 2+ affinity chromatography followed by Protein A chromatography. SDS-PAGE analysis demonstrated that CD147 was of high purity and indicated that there was no residual Fc fragment or 3C protease. Lane 1, recombinant eukaryotic-expressed CD147-Fc, which has a dimer formation and has been partially degraded; Lane 2, the recombinant CD147-Fc protein was treated with HRV 3C protease for 16 h, resulting in a mixture of CD147-Fc, CD147, Fc fragments and HRV 3C protease; Lane 3, mixture purified by Ni 2+ affinity chromatography, which is not very powerful, and a certain amount of recombinant protein and HRV 3C protease remain; Lane 4, CD147 immunogen purified by Protein A chromatography, no recombinant protein or HRV 3C protease was detected; Lane M, protein molecular weight marker. HRV, human rhinovirus.

    Journal: Molecular Medicine Reports

    Article Title: Purification of a polyclonal antibody against CD147 for ELISA using antigen-immunoaffinity chromatography

    doi: 10.3892/mmr.2017.6523

    Figure Lengend Snippet: SDS-PAGE analysis of the CD147 antigen prior to rabbit immunization. Eukaryotic-expressed recombinant protein CD147-Fc was treated with HRV 3C protease for 16 h, and the mixture was purified by Ni 2+ affinity chromatography followed by Protein A chromatography. SDS-PAGE analysis demonstrated that CD147 was of high purity and indicated that there was no residual Fc fragment or 3C protease. Lane 1, recombinant eukaryotic-expressed CD147-Fc, which has a dimer formation and has been partially degraded; Lane 2, the recombinant CD147-Fc protein was treated with HRV 3C protease for 16 h, resulting in a mixture of CD147-Fc, CD147, Fc fragments and HRV 3C protease; Lane 3, mixture purified by Ni 2+ affinity chromatography, which is not very powerful, and a certain amount of recombinant protein and HRV 3C protease remain; Lane 4, CD147 immunogen purified by Protein A chromatography, no recombinant protein or HRV 3C protease was detected; Lane M, protein molecular weight marker. HRV, human rhinovirus.

    Article Snippet: The Fc fragment was cleaved by Human Rhinovirus (HRV) 3C Protease (Sino Biological, Inc., Beijing, China).

    Techniques: SDS Page, Recombinant, Purification, Affinity Chromatography, Chromatography, Molecular Weight, Marker