bhk-21 cells Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    ATCC baby hamster kidney fibroblasts bhk 21 cells
    IBV S1 inhibits rcMBLs antiviral activity . <t>BHK-21</t> cells were infected with IBV Beaudette at an MOI of 0.1 in the presence or absence of 10 μg/ml rcMBL and 50 μg/ml M41 S1. Infection was determined by immunofluorescence using an antibody directed against the IBV nucleocapsid protein at 8 h post-infection.
    Baby Hamster Kidney Fibroblasts Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney fibroblasts bhk 21 cells/product/ATCC
    Average 99 stars, based on 32 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney fibroblasts bhk 21 cells - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    90
    Millipore baby hamster kidney bhk 21 cells
    Quantification of pGFP-PAC replicon GFP expression over time in the presence and absence of aptamer. <t>BHK-21</t> cells were seeded and maintained for 24 h in 10 % FCS/DMEM at 37 °C, 5 % CO 2 prior to transfection with 1 µg pGFP-PAC replicon RNA. GFP expression was monitored using an IncuCyte FLR microscope. Data are presented as mean green object count of the pGFP-PAC replicon over up to 12 h from the point at which GFP expression is first detected. (a) GFP expression with 2′F-Cy3-47tr, 2′F-Cy3-52tr or cRNA at final concentrations of 500 nM. GFP expression with increasing concentrations (0 to 500 nM) of 2′F-Cy3-47tr (b) or 2′F-Cy3-52tr (c). (d) 2′-CMC (0–50 µM). Shown is the mean object count from a collection of nine images, allowing se to be calculated.
    Baby Hamster Kidney Bhk 21 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney bhk 21 cells/product/Millipore
    Average 90 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney bhk 21 cells - by Bioz Stars, 2020-08
    90/100 stars
      Buy from Supplier

    98
    ATCC baby hamster kidney cells bhk 21 cells
    Quantification of pGFP-PAC replicon GFP expression over time in the presence and absence of aptamer. <t>BHK-21</t> cells were seeded and maintained for 24 h in 10 % FCS/DMEM at 37 °C, 5 % CO 2 prior to transfection with 1 µg pGFP-PAC replicon RNA. GFP expression was monitored using an IncuCyte FLR microscope. Data are presented as mean green object count of the pGFP-PAC replicon over up to 12 h from the point at which GFP expression is first detected. (a) GFP expression with 2′F-Cy3-47tr, 2′F-Cy3-52tr or cRNA at final concentrations of 500 nM. GFP expression with increasing concentrations (0 to 500 nM) of 2′F-Cy3-47tr (b) or 2′F-Cy3-52tr (c). (d) 2′-CMC (0–50 µM). Shown is the mean object count from a collection of nine images, allowing se to be calculated.
    Baby Hamster Kidney Cells Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney cells bhk 21 cells/product/ATCC
    Average 98 stars, based on 21 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney cells bhk 21 cells - by Bioz Stars, 2020-08
    98/100 stars
      Buy from Supplier

    92
    Roche bhk 21 cells
    In vitro ). TR, terminal repeats. (b, c) Detection of viral proteins in infected <t>BHK-21</t> (3 PFU/cell, 6 h) cellular lysates using Western blotting (WB). kLANA was detected with MAb LN53, and mLANA was detected with MAb 6A3. Blotting against the MuHV-4 M3 protein and actin was used to assess levels of infection and loading, respectively. In panel b, expression of vCyclin and YFP was confirmed with anti-vCyclin and anti-GFP immunoblotting, respectively. (d) Images (confocal slices) depicting localization of LANA proteins in BHK-21 cells infected as described for panel a. DNA was stained with DAPI. Bar, 10 μm. (e) Relative levels (ΔΔ C T ) of M11, M3, and ORF63 in infected BHK-21 cells (5 PFU/cell, 8 h) compared to uninfected cells, normalized to GAPDH. Bars indicate mean fold changes ± standard deviations (SD). (f) Growth curves in BHK-21 cells infected with 0.01 PFU/cell. Total virus titers were determined. Time zero indicates input of virus after washing inoculum. Virus titers did not differ significantly between infection groups (Kruskal-Wallis test).
    Bhk 21 Cells, supplied by Roche, used in various techniques. Bioz Stars score: 92/100, based on 518 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/Roche
    Average 92 stars, based on 518 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Bio-Rad bhk 21 cells
    Immunofluorescence staining of protein E in <t>BHK-21</t> cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein
    Bhk 21 Cells, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 92/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/Bio-Rad
    Average 92 stars, based on 517 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    92
    Thermo Fisher baby hamster kidney bhk 21 cells
    Effect of blocking de novo synthesis of PI(4,5)P 2 with 1-butanol on FMDV and VSV internalization. (A) <t>BHK-21</t> cells transfected (24 h) with PH-PLC-eGFP (green) were treated or not with 1.5% 1-butanol or 2-butanol for 5 min and then fixed and observed by confocal microscopy. Nuclei were stained using ToPro-3 (blue). Bar 10 µm. (B) Treatment with 1-butanol inhibits clathrin-dependent endocytosis. BHK-21 cells were treated as in (A) were incubated with fluorescent TF and processed as described in the legend of Fig. 1 . Bar: 10 µm. (C) Reduction of the ability of cells to internalize FMDV and VSV upon 1-butanol treatment. Cells treated as in (A) were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min in the presence of the drugs. Bars represent the mean percentage of cells with internalized virions ± SD, normalized to the level of cells with internalized virions in control samples. At least 500 cells per coverslip were scored for each case (3 coverslips). Asterisks denote statistically significant differences (ANOVA P≤0.05).
    Baby Hamster Kidney Bhk 21 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 222 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney bhk 21 cells/product/Thermo Fisher
    Average 92 stars, based on 222 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney bhk 21 cells - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    93
    DSMZ baby hamster kidney 21 bhk 21 cells
    Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. <t>BHK-21</t> cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.
    Baby Hamster Kidney 21 Bhk 21 Cells, supplied by DSMZ, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney 21 bhk 21 cells/product/DSMZ
    Average 93 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney 21 bhk 21 cells - by Bioz Stars, 2020-08
    93/100 stars
      Buy from Supplier

    91
    BIO-CAT bhk 21 cells
    Testing the interaction between Mtk and SdhB in <t>BHK-21</t> cells. The interaction between Mtk and SdhB was tested using the F2H assay 24 h after the transfection of BHK-21 cells. ( a ) DAPI channel. ( b ) GFP channel. ( c ) RFP channel. ( d ) Merged. The presence of both green and red spots (arrows) in the GFP and RFP channels, respectively, shows the interaction between Mtk and SdhB as confirmed by the presence of both green and red spots. Scale bars = 25 µm.
    Bhk 21 Cells, supplied by BIO-CAT, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/BIO-CAT
    Average 91 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    85
    ATCC bhk 21 cells
    S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on <t>BHK-21</t> cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.
    Bhk 21 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 85/100, based on 2862 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bhk 21 cells/product/ATCC
    Average 85 stars, based on 2862 article reviews
    Price from $9.99 to $1999.99
    bhk 21 cells - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    91
    KeraFAST baby hamster kidney bhk 21 cells
    S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on <t>BHK-21</t> cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.
    Baby Hamster Kidney Bhk 21 Cells, supplied by KeraFAST, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/baby hamster kidney bhk 21 cells/product/KeraFAST
    Average 91 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    baby hamster kidney bhk 21 cells - by Bioz Stars, 2020-08
    91/100 stars
      Buy from Supplier

    85
    Boster Bio transfections baby hamster kidney bhk 21 cells
    Detection of the FMDV structure protein and FMDV capsid. Immunofluoresence was used to determine the expression levels of the FMDV structure protein following <t>transfection</t> with either pcDNA3.1(+) (a), pA (b) pB (c) or pC (d). FMDV capsid was observed by TEM of <t>BHK-21</t> cells transfected with pA (e) or pC (d).
    Transfections Baby Hamster Kidney Bhk 21 Cells, supplied by Boster Bio, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/transfections baby hamster kidney bhk 21 cells/product/Boster Bio
    Average 85 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    transfections baby hamster kidney bhk 21 cells - by Bioz Stars, 2020-08
    85/100 stars
      Buy from Supplier

    Image Search Results


    IBV S1 inhibits rcMBLs antiviral activity . BHK-21 cells were infected with IBV Beaudette at an MOI of 0.1 in the presence or absence of 10 μg/ml rcMBL and 50 μg/ml M41 S1. Infection was determined by immunofluorescence using an antibody directed against the IBV nucleocapsid protein at 8 h post-infection.

    Journal: Virology

    Article Title: Chicken mannose binding lectin has antiviral activity towards infectious bronchitis virus

    doi: 10.1016/j.virol.2017.06.028

    Figure Lengend Snippet: IBV S1 inhibits rcMBLs antiviral activity . BHK-21 cells were infected with IBV Beaudette at an MOI of 0.1 in the presence or absence of 10 μg/ml rcMBL and 50 μg/ml M41 S1. Infection was determined by immunofluorescence using an antibody directed against the IBV nucleocapsid protein at 8 h post-infection.

    Article Snippet: 2.1 Cell lines Human Embryonic Kidney (HEK) 293 T cells, Baby Hamster Kidney fibroblasts (BHK) 21 cells (ATCC CCL-10), and Human Cervical adenocarcinoma (HeLa) R19 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% Fetal Calf Serum (FCS) (Bodinco BV, The Netherlands), containing penicillin (100 units/ml) and streptomycin (100 µg/ml) (Gibco).

    Techniques: Activity Assay, Infection, Immunofluorescence

    rcMBL inhibits IBV-Beaudette infection of BHK 21 cells in a concentration dependent manner . BHK-21 cells were inoculated after pre-incubation of IBV-Beaudette (MOI of 0.1) with various concentrations of rcMBL. A) Infection was determined by immunofluorescence using an antibody directed against the IBV nucleocapsid protein at 8 h post-infection. Bar: 50 µm. B) Infection was determined by quantitative PCR: the IBV viral genome expression relative to GAPDH gene expression was determined and expressed relative to the no-rcMBL control. C) The toxic effects of rcMBL on BHK-21 cells was determined by the WST-1 assay. Asterisks indicate statistical significant difference compared to the no rcMBL control. Shown are mean ± SEM of three independent experiments.

    Journal: Virology

    Article Title: Chicken mannose binding lectin has antiviral activity towards infectious bronchitis virus

    doi: 10.1016/j.virol.2017.06.028

    Figure Lengend Snippet: rcMBL inhibits IBV-Beaudette infection of BHK 21 cells in a concentration dependent manner . BHK-21 cells were inoculated after pre-incubation of IBV-Beaudette (MOI of 0.1) with various concentrations of rcMBL. A) Infection was determined by immunofluorescence using an antibody directed against the IBV nucleocapsid protein at 8 h post-infection. Bar: 50 µm. B) Infection was determined by quantitative PCR: the IBV viral genome expression relative to GAPDH gene expression was determined and expressed relative to the no-rcMBL control. C) The toxic effects of rcMBL on BHK-21 cells was determined by the WST-1 assay. Asterisks indicate statistical significant difference compared to the no rcMBL control. Shown are mean ± SEM of three independent experiments.

    Article Snippet: 2.1 Cell lines Human Embryonic Kidney (HEK) 293 T cells, Baby Hamster Kidney fibroblasts (BHK) 21 cells (ATCC CCL-10), and Human Cervical adenocarcinoma (HeLa) R19 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% Fetal Calf Serum (FCS) (Bodinco BV, The Netherlands), containing penicillin (100 units/ml) and streptomycin (100 µg/ml) (Gibco).

    Techniques: Infection, Concentration Assay, Incubation, Immunofluorescence, Real-time Polymerase Chain Reaction, Expressing, WST-1 Assay

    Preimmunization of rNS1 and rDR4 suppressed elicitation of neutralizing anti-DENV EIII titers. Experimental mice were firstly immunized with adjuvant (without mixed with proteins), rGST, rNS1, rTACI and rDR4 for 2 cycles, and then immunization with rEIII for additional 2 cycles ( A ) (experiment outline). The anti-EIII Ig titers were analyzed using ELISA in the 5 th week ( B ). The neutralizing properties of these anti-EIII Ig were analyzed by protection of BHK-21 cells against DENV infection in vitro . The cell survival rates were recorded after 96 h challenges of DENV (MOI = 0.5) and 96 h treatments of heat-inactivated anti-serum (20 µL/well of microtiter plate) from respective groups ( C ). * P

    Journal: Scientific Reports

    Article Title: Suppressed humoral immunity is associated with dengue nonstructural protein NS1-elicited anti-death receptor antibody fractions in mice

    doi: 10.1038/s41598-020-62958-0

    Figure Lengend Snippet: Preimmunization of rNS1 and rDR4 suppressed elicitation of neutralizing anti-DENV EIII titers. Experimental mice were firstly immunized with adjuvant (without mixed with proteins), rGST, rNS1, rTACI and rDR4 for 2 cycles, and then immunization with rEIII for additional 2 cycles ( A ) (experiment outline). The anti-EIII Ig titers were analyzed using ELISA in the 5 th week ( B ). The neutralizing properties of these anti-EIII Ig were analyzed by protection of BHK-21 cells against DENV infection in vitro . The cell survival rates were recorded after 96 h challenges of DENV (MOI = 0.5) and 96 h treatments of heat-inactivated anti-serum (20 µL/well of microtiter plate) from respective groups ( C ). * P

    Article Snippet: Baby hamster kidney BHK-21 cells (ATCC® CCL-10) were maintained in DMEM 10% FBS, 4.5 g/L glucose, 6 mM glutamine cell culture medium and were used in the analysis of anti-envelop EIII protein neutralizing antibody.

    Techniques: Mouse Assay, Enzyme-linked Immunosorbent Assay, Infection, In Vitro

    D471G substitution affects the ability of LCMV-NP to promote replication and gene expression of an LCMV MG. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, together with 0.6 μg of pC-L, 0.3 μg of pC wt or mutant NPs HA-tagged, and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies At 48 hpt, MG driven GFP expression (A) and luciferase activity in TCS (B) were determined. Cell lysates were used to determine expression levels of wt and mutant NPs by WB using an anti-HA antibody (C). GAPDH was used as a loading control. Empty pC was used as a negative control. Percentages of relative luciferase units (% RLUs) were normalized with respect to the activity of the wt NP, after normalization of transfection efficiencies based on Cluc luminescence values. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane as described in material and methods.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: D471G substitution affects the ability of LCMV-NP to promote replication and gene expression of an LCMV MG. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, together with 0.6 μg of pC-L, 0.3 μg of pC wt or mutant NPs HA-tagged, and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies At 48 hpt, MG driven GFP expression (A) and luciferase activity in TCS (B) were determined. Cell lysates were used to determine expression levels of wt and mutant NPs by WB using an anti-HA antibody (C). GAPDH was used as a loading control. Empty pC was used as a negative control. Percentages of relative luciferase units (% RLUs) were normalized with respect to the activity of the wt NP, after normalization of transfection efficiencies based on Cluc luminescence values. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane as described in material and methods.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Expressing, Transfection, Mutagenesis, Plasmid Preparation, Luciferase, Activity Assay, Western Blot, Negative Control

    Effects of D471 substitution on LCMV-NP functions. (A) NP-NP interaction: Human 293T cells were co-transfected as described in Figure 1 B with 2 μg of the indicated pC wt or mutant NP-VP16 fusion proteins, together with 2 μg of NP-GAL4 expression plasmids. At 72 hpt, cell extracts were prepared to determine the strength of the interaction. VP16 expression plasmid was used as negative control. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-NP-GAL4) after normalization of transfection efficiencies based on levels of Renilla luciferase activity driven by plasmid pRL SV40. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (B) NP-Z interaction: Human 293T cells were co-transfected as described in Figure 2 B with 2 μg of the indicated pC wt or mutant NP-VP16, together with 2 μg of GAL4-Z expression plasmids. At 48 hpt, cell extracts were prepared to determine the strength of the interaction and protein expression. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-GAL4-Z) after normalization of transfection efficiencies based on Renilla luciferase values. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (C) Replication and transcription activity: BHK-21 cells were co-transfected with the LCMV MG as described in Figure 3 together with expression plasmids for the viral polymerase (L) and wt or indicated mutant NPs, and pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, TCS were collected for luciferase assay and cell lysates were prepared for protein detection. Empty pC was used as a negative control. RLUs (%) were calculated based on the replication and transcription activity mediated by wt NP, after normalization by Cluc luminescence values. Expression levels of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control. (D) Inhibition of induction of IFN-I: Human 293T cells were co-transfected as described in Figure 4 with 0.1 μg of the indicated pC-NP HA-tagged expression vectors together with the IFNβ reporter plasmids. At 16 hpt, cells were infected with SeV (moi=3) to induce activation of the IFNβ promoter, and 24 hours later cell lysates were prepared for luciferase assay and detection of protein expression. Luciferase values were normalized with respect to those obtained in cells transfected with Empty pC and infected with SeV, after adjusting for renilla luminescence values. Expression of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: Effects of D471 substitution on LCMV-NP functions. (A) NP-NP interaction: Human 293T cells were co-transfected as described in Figure 1 B with 2 μg of the indicated pC wt or mutant NP-VP16 fusion proteins, together with 2 μg of NP-GAL4 expression plasmids. At 72 hpt, cell extracts were prepared to determine the strength of the interaction. VP16 expression plasmid was used as negative control. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-NP-GAL4) after normalization of transfection efficiencies based on levels of Renilla luciferase activity driven by plasmid pRL SV40. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (B) NP-Z interaction: Human 293T cells were co-transfected as described in Figure 2 B with 2 μg of the indicated pC wt or mutant NP-VP16, together with 2 μg of GAL4-Z expression plasmids. At 48 hpt, cell extracts were prepared to determine the strength of the interaction and protein expression. Reporter gene activation (FFL) is shown as percentage of wt interaction (pC-NP-VP16 and pC-GAL4-Z) after normalization of transfection efficiencies based on Renilla luciferase values. Cell lysates were used to detect expression of wt and mutant NPs by WB using an anti-VP16 polyclonal antibody. GAPDH was used as a loading control. (C) Replication and transcription activity: BHK-21 cells were co-transfected with the LCMV MG as described in Figure 3 together with expression plasmids for the viral polymerase (L) and wt or indicated mutant NPs, and pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, TCS were collected for luciferase assay and cell lysates were prepared for protein detection. Empty pC was used as a negative control. RLUs (%) were calculated based on the replication and transcription activity mediated by wt NP, after normalization by Cluc luminescence values. Expression levels of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control. (D) Inhibition of induction of IFN-I: Human 293T cells were co-transfected as described in Figure 4 with 0.1 μg of the indicated pC-NP HA-tagged expression vectors together with the IFNβ reporter plasmids. At 16 hpt, cells were infected with SeV (moi=3) to induce activation of the IFNβ promoter, and 24 hours later cell lysates were prepared for luciferase assay and detection of protein expression. Luciferase values were normalized with respect to those obtained in cells transfected with Empty pC and infected with SeV, after adjusting for renilla luminescence values. Expression of wt and mutant NPs were determined by WB using an anti-HA antibody. GAPDH was used as a loading control.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Transfection, Mutagenesis, Expressing, Plasmid Preparation, Negative Control, Activation Assay, Luciferase, Activity Assay, Western Blot, Inhibition, Infection

    Dominant negative effect of LCMV-NP D471G on viral replication and transcription. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, 0.6 μg of pC-L, and the indicated amounts (ng) of pC-LCMV-NP HA-tagged (NP); together with empty pC (Empty) or pC LCMV-NP D471G HA-tagged (D471G), and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, MG activity was determined by GFP expression (A) and luciferase activity from TCS (B). Reporter gene activation is shown as induction over an empty pC vector-transfected control.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: Dominant negative effect of LCMV-NP D471G on viral replication and transcription. BHK-21 cells were co-transfected in triplicate with 0.5 μg of pPolI GFP-Pur/Gluc, 0.6 μg of pC-L, and the indicated amounts (ng) of pC-LCMV-NP HA-tagged (NP); together with empty pC (Empty) or pC LCMV-NP D471G HA-tagged (D471G), and 0.1μg of the pSV40-Cluc expression vector to normalize transfection efficiencies. At 48 hpt, MG activity was determined by GFP expression (A) and luciferase activity from TCS (B). Reporter gene activation is shown as induction over an empty pC vector-transfected control.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Dominant Negative Mutation, Transfection, Expressing, Plasmid Preparation, Activity Assay, Luciferase, Activation Assay

    Dominant negative effect of NP D471G on LCMV infection. (A) Characterization of stable cell lines expressing HA-tagged wt mutant or D471G NPs. Parental and NP-HA expressing BHK-21 cell lines were examined by immunofluorescence with an anti-HA (α-HA) antibody (NP staining). Cellular nuclei were stained with DAPI. Representative merged images are illustrated. Cells lysates were prepared and 100 μg of total cellular protein content were analyzed by WB using an anti-HA antibody (B). GAPDH was used as a loading control. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane, as described in material and methods. Kinetics of LCMV (C) and VSV-GFP (D) propagation: Parental and NP-HA expressing BHK-21 cell lines were infected with LCMV (moi = 0.01) or VSV-GFP (moi = 0.001) in triplicates. TCS at the indicated hpi were titrated using a focus forming unit assay on Vero cells as described in materials and methods. Dashed lines indicate the limit of detection for the assay.

    Journal: Viruses

    Article Title: D471G Mutation in LCMV-NP Affects its Ability to Self-associate and Results in a Dominant Negative Effect in Viral RNA Synthesis

    doi: 10.3390/v4102137

    Figure Lengend Snippet: Dominant negative effect of NP D471G on LCMV infection. (A) Characterization of stable cell lines expressing HA-tagged wt mutant or D471G NPs. Parental and NP-HA expressing BHK-21 cell lines were examined by immunofluorescence with an anti-HA (α-HA) antibody (NP staining). Cellular nuclei were stained with DAPI. Representative merged images are illustrated. Cells lysates were prepared and 100 μg of total cellular protein content were analyzed by WB using an anti-HA antibody (B). GAPDH was used as a loading control. Numbers at the bottom of each WB lane represent the quantification of band intensities normalized to wt NP lane, as described in material and methods. Kinetics of LCMV (C) and VSV-GFP (D) propagation: Parental and NP-HA expressing BHK-21 cell lines were infected with LCMV (moi = 0.01) or VSV-GFP (moi = 0.001) in triplicates. TCS at the indicated hpi were titrated using a focus forming unit assay on Vero cells as described in materials and methods. Dashed lines indicate the limit of detection for the assay.

    Article Snippet: Cells and Viruses Baby hamster kidney (BHK-21) cells (ATCC CCL-10) and human embryonic kidney (293T) cells (ATCC CRL-11268) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), L-glutamine (2mM), penicillin (100 units/ml) and streptomycin (100μg/ml), in a 5 % CO2 atmosphere at 37°C [ , ].

    Techniques: Dominant Negative Mutation, Infection, Stable Transfection, Expressing, Mutagenesis, Immunofluorescence, Staining, Western Blot

    Staining of rabies virus granules in BHK-21 cells 4 days after transfection. (A, C, E) Cells transfected with rCTN-Gluc, rCTN/G Q333R -Gluc and rCTNΔG-Gluc plus pVAX1-N, P, L. (B, D, F) Cells transfected with rCTN-Gluc, rCTN/G Q333R -Gluc and rCTNΔG-Gluc plus pVAX1-N, P, G, L. (G–H) Negative control.

    Journal: Virus Research

    Article Title: Development of recombinant rabies viruses vectors with Gaussia luciferase reporter based on Chinese vaccine strain CTN181

    doi: 10.1016/j.virusres.2011.05.018

    Figure Lengend Snippet: Staining of rabies virus granules in BHK-21 cells 4 days after transfection. (A, C, E) Cells transfected with rCTN-Gluc, rCTN/G Q333R -Gluc and rCTNΔG-Gluc plus pVAX1-N, P, L. (B, D, F) Cells transfected with rCTN-Gluc, rCTN/G Q333R -Gluc and rCTNΔG-Gluc plus pVAX1-N, P, G, L. (G–H) Negative control.

    Article Snippet: 2.1 Cells culture Baby hamster kidney (BHK-21) cells (ATCC, Rockville, MD) and mouse neuroblastoma (MNA) cells (kindly provided by Wuhan Institute of Biologic Product, Wuhan, China) were maintained in Dulbecco's modified Eagle's medium (D-MEM) containing 10% heat-inactivated fetal bovine serum (FBS) (Gibco-BRL, Gaithersburg, MD) at 37 °C in a humidified 5% CO2 atmosphere.

    Techniques: Staining, Transfection, Negative Control

    Interaction of BHK cells with PMMA–PTE nanoparticles at a concentration of 25 ppm. The images represent the nucleus stained with DAPI (A), cellular matrix stained with nanoparticles (B), and an overlay of the two images (C), with the magnified images in the inset showing nanoparticle distribution inside the cells (D). Magnified view (D) of nucleus stained with DAPI (i), cellular matrix stained with nanoparticles (ii), cells observed under DIC mode (iii), and overlay of (i)–(iv). The cells were imaged at a magnification of 60× oil objective using a confocal microscope (Olympus FV1000).

    Journal: ACS Omega

    Article Title: Controlled Dye Aggregation in Sodium Dodecylsulfate-Stabilized Poly(methylmethacrylate) Nanoparticles as Fluorescent Imaging Probes

    doi: 10.1021/acsomega.8b00785

    Figure Lengend Snippet: Interaction of BHK cells with PMMA–PTE nanoparticles at a concentration of 25 ppm. The images represent the nucleus stained with DAPI (A), cellular matrix stained with nanoparticles (B), and an overlay of the two images (C), with the magnified images in the inset showing nanoparticle distribution inside the cells (D). Magnified view (D) of nucleus stained with DAPI (i), cellular matrix stained with nanoparticles (ii), cells observed under DIC mode (iii), and overlay of (i)–(iv). The cells were imaged at a magnification of 60× oil objective using a confocal microscope (Olympus FV1000).

    Article Snippet: Internalization of Nanoparticles into Mammalian Cells for Imaging To establish a cellular model system, BHK cells (ATCC CCL-10) were used to understand the uptake and internalization of the dye-encapsulated polymer nanoparticles.

    Techniques: Concentration Assay, Staining, Microscopy

    (A) Specific typing of clinical DENV isolates by RT-PCR of cytoplasmic RNAs of virus-infected and uninfected BHK-21 cells. Agarose gel electrophoresis of RT-PCR products displaying diagnostic target sizes of 169, 362, 265 and 426 base-pairs (bp) for DENV-1, -2, -3 and -4, respectively, which were absent for Chikungunya virus-infected (CHKV) and uninfected BHK-21 cells (Control), thus showing good specificity. The 100-bp DNA ladder size marker was also included. (B) Normalized impedance signal response as a function of viral infection time for four different DENV serotypes isolated from clinical samples. DENV-2 NGC-infected cells and uninfected control cells were also included. EIS data were recorded at an open circuit potential, frequency of 4 kHz, and excitation amplitude of 10 mV. Vertical lines for each virus indicate the onset of CPE. Conditions: MOI=5, BHK-21 host cells; growth medium constitutes the background electrolyte.

    Journal: Biosensors & Bioelectronics

    Article Title: Impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses

    doi: 10.1016/j.bios.2015.03.018

    Figure Lengend Snippet: (A) Specific typing of clinical DENV isolates by RT-PCR of cytoplasmic RNAs of virus-infected and uninfected BHK-21 cells. Agarose gel electrophoresis of RT-PCR products displaying diagnostic target sizes of 169, 362, 265 and 426 base-pairs (bp) for DENV-1, -2, -3 and -4, respectively, which were absent for Chikungunya virus-infected (CHKV) and uninfected BHK-21 cells (Control), thus showing good specificity. The 100-bp DNA ladder size marker was also included. (B) Normalized impedance signal response as a function of viral infection time for four different DENV serotypes isolated from clinical samples. DENV-2 NGC-infected cells and uninfected control cells were also included. EIS data were recorded at an open circuit potential, frequency of 4 kHz, and excitation amplitude of 10 mV. Vertical lines for each virus indicate the onset of CPE. Conditions: MOI=5, BHK-21 host cells; growth medium constitutes the background electrolyte.

    Article Snippet: 2.3 Culturing and maintenance of BHK-21 cells BHK-21 cells (American Type Culture Collection) were cultured in a T25 tissue culture flask containing 3 mL of growth medium (RPMI-1640 medium supplemented with 10% FBS and 1×penicillin–streptomycin solution).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Agarose Gel Electrophoresis, Diagnostic Assay, Marker, Isolation, Impedance Spectroscopy

    Microscopic images of (A) BHK-21 cells after 5 days of infection with DENV-2 NGC at MOI=1, and (B) control uninfected BHK-21 cells after 5 days of incubation with growth medium. Scale bar=100 µm.

    Journal: Biosensors & Bioelectronics

    Article Title: Impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses

    doi: 10.1016/j.bios.2015.03.018

    Figure Lengend Snippet: Microscopic images of (A) BHK-21 cells after 5 days of infection with DENV-2 NGC at MOI=1, and (B) control uninfected BHK-21 cells after 5 days of incubation with growth medium. Scale bar=100 µm.

    Article Snippet: 2.3 Culturing and maintenance of BHK-21 cells BHK-21 cells (American Type Culture Collection) were cultured in a T25 tissue culture flask containing 3 mL of growth medium (RPMI-1640 medium supplemented with 10% FBS and 1×penicillin–streptomycin solution).

    Techniques: Infection, Incubation

    (A) EIS Bode diagram recorded in a frequency range of 100 Hz to 1 MHz shows the impedance signal response of the BHK-21 cell-based biosensor measured prior to infection (•), and during day 5 of infection (♦) with DENV-2 (NGC strain) at MOI=5. (B) BHK-21 cell-based biosensor impedance signal as a function of DENV-2 NGC infection time at different MOI (0, 0.1, 1, 5, 10). Impedance signal response in the presence of virus ( Z virus ) was normalized against the impedance signal response derived from the same biosensor in the absence of virus at 0 hpi ( Z virus =0). EIS data were recorded at open circuit potential, frequency of 4 kHz, and excitation amplitude of 10 mV. Vertical lines for each MOI indicate the onset of CPE. (C) Plot of CIT 50 against the logarithm of virus titer (MOI). CIT 50 refers to the time taken for 50% reduction in cell impedance.

    Journal: Biosensors & Bioelectronics

    Article Title: Impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses

    doi: 10.1016/j.bios.2015.03.018

    Figure Lengend Snippet: (A) EIS Bode diagram recorded in a frequency range of 100 Hz to 1 MHz shows the impedance signal response of the BHK-21 cell-based biosensor measured prior to infection (•), and during day 5 of infection (♦) with DENV-2 (NGC strain) at MOI=5. (B) BHK-21 cell-based biosensor impedance signal as a function of DENV-2 NGC infection time at different MOI (0, 0.1, 1, 5, 10). Impedance signal response in the presence of virus ( Z virus ) was normalized against the impedance signal response derived from the same biosensor in the absence of virus at 0 hpi ( Z virus =0). EIS data were recorded at open circuit potential, frequency of 4 kHz, and excitation amplitude of 10 mV. Vertical lines for each MOI indicate the onset of CPE. (C) Plot of CIT 50 against the logarithm of virus titer (MOI). CIT 50 refers to the time taken for 50% reduction in cell impedance.

    Article Snippet: 2.3 Culturing and maintenance of BHK-21 cells BHK-21 cells (American Type Culture Collection) were cultured in a T25 tissue culture flask containing 3 mL of growth medium (RPMI-1640 medium supplemented with 10% FBS and 1×penicillin–streptomycin solution).

    Techniques: Impedance Spectroscopy, Infection, Derivative Assay

    Control experiments to compare biosensor impedance signal responses of Vero (--- --- ---), BHK-21 ( — ), and C6/36 (•••) cells toward DENV-2 NGC infection. EIS data were recorded at open circuit potential, frequency of 4 kHz, and excitation amplitude of 10 mV. Conditions: infection with DENV-2 (NGC strain); MOI=5; growth medium constitutes the background electrolyte.

    Journal: Biosensors & Bioelectronics

    Article Title: Impedimetric cell-based biosensor for real-time monitoring of cytopathic effects induced by dengue viruses

    doi: 10.1016/j.bios.2015.03.018

    Figure Lengend Snippet: Control experiments to compare biosensor impedance signal responses of Vero (--- --- ---), BHK-21 ( — ), and C6/36 (•••) cells toward DENV-2 NGC infection. EIS data were recorded at open circuit potential, frequency of 4 kHz, and excitation amplitude of 10 mV. Conditions: infection with DENV-2 (NGC strain); MOI=5; growth medium constitutes the background electrolyte.

    Article Snippet: 2.3 Culturing and maintenance of BHK-21 cells BHK-21 cells (American Type Culture Collection) were cultured in a T25 tissue culture flask containing 3 mL of growth medium (RPMI-1640 medium supplemented with 10% FBS and 1×penicillin–streptomycin solution).

    Techniques: Infection, Impedance Spectroscopy

    Quantification of pGFP-PAC replicon GFP expression over time in the presence and absence of aptamer. BHK-21 cells were seeded and maintained for 24 h in 10 % FCS/DMEM at 37 °C, 5 % CO 2 prior to transfection with 1 µg pGFP-PAC replicon RNA. GFP expression was monitored using an IncuCyte FLR microscope. Data are presented as mean green object count of the pGFP-PAC replicon over up to 12 h from the point at which GFP expression is first detected. (a) GFP expression with 2′F-Cy3-47tr, 2′F-Cy3-52tr or cRNA at final concentrations of 500 nM. GFP expression with increasing concentrations (0 to 500 nM) of 2′F-Cy3-47tr (b) or 2′F-Cy3-52tr (c). (d) 2′-CMC (0–50 µM). Shown is the mean object count from a collection of nine images, allowing se to be calculated.

    Journal: The Journal of General Virology

    Article Title: Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

    doi: 10.1099/vir.0.067751-0

    Figure Lengend Snippet: Quantification of pGFP-PAC replicon GFP expression over time in the presence and absence of aptamer. BHK-21 cells were seeded and maintained for 24 h in 10 % FCS/DMEM at 37 °C, 5 % CO 2 prior to transfection with 1 µg pGFP-PAC replicon RNA. GFP expression was monitored using an IncuCyte FLR microscope. Data are presented as mean green object count of the pGFP-PAC replicon over up to 12 h from the point at which GFP expression is first detected. (a) GFP expression with 2′F-Cy3-47tr, 2′F-Cy3-52tr or cRNA at final concentrations of 500 nM. GFP expression with increasing concentrations (0 to 500 nM) of 2′F-Cy3-47tr (b) or 2′F-Cy3-52tr (c). (d) 2′-CMC (0–50 µM). Shown is the mean object count from a collection of nine images, allowing se to be calculated.

    Article Snippet: Complete Dulbecco’s modified Eagle’s medium (DMEM) was removed from the BHK-21 cells and replaced with the ESCORT reagent mixture (Sigma-Aldrich).

    Techniques: Expressing, Transfection, Microscopy

    Aptamers can be detected in BHK-21 cells. (a) Cells grown on glass coverslips were transfected with 50 nM 2′F-Cy3-47tr or 2′F-Cy3-52tr aptamer (red). At 24 h post-transfection, cells were fixed, stained (DAPI) and imaged using an upright confocal microscope (Zeiss). Bars, 10 µm. (b, c) Cells were seeded and maintained for up to 24 h in 10 % FCS/DMEM at 37 °C, 5 % CO 2 prior to transfection with 1 µg pGFP-PAC replicon RNA in the presence or absence of 2′F-Cy3-47tr (b) or 2′F-Cy3-52tr (c) at final concentrations of 500 nM. An IncuCyte was used to take nine images of every well, every 30 min. A selection of images at t = 2, 4, 8 and 12 h post-transfection are shown, as a merge of fluorescent and phase-contrast images, with mock-treated cells as a control.

    Journal: The Journal of General Virology

    Article Title: Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

    doi: 10.1099/vir.0.067751-0

    Figure Lengend Snippet: Aptamers can be detected in BHK-21 cells. (a) Cells grown on glass coverslips were transfected with 50 nM 2′F-Cy3-47tr or 2′F-Cy3-52tr aptamer (red). At 24 h post-transfection, cells were fixed, stained (DAPI) and imaged using an upright confocal microscope (Zeiss). Bars, 10 µm. (b, c) Cells were seeded and maintained for up to 24 h in 10 % FCS/DMEM at 37 °C, 5 % CO 2 prior to transfection with 1 µg pGFP-PAC replicon RNA in the presence or absence of 2′F-Cy3-47tr (b) or 2′F-Cy3-52tr (c) at final concentrations of 500 nM. An IncuCyte was used to take nine images of every well, every 30 min. A selection of images at t = 2, 4, 8 and 12 h post-transfection are shown, as a merge of fluorescent and phase-contrast images, with mock-treated cells as a control.

    Article Snippet: Complete Dulbecco’s modified Eagle’s medium (DMEM) was removed from the BHK-21 cells and replaced with the ESCORT reagent mixture (Sigma-Aldrich).

    Techniques: Transfection, Staining, Microscopy, Selection

    Effect of aptamers on translation. BHK-21 cells were transfected with a construct containing Renilla and firefly luciferase reporter genes under either (a) cap- or (b) FMDV IRES-mediated translation respectively (pRFMDVF). At 24 h post-transfection of the constructs, cells were subjected to a second transfection with aptamers 2′F-Cy3-47tr, 2′F-Cy3-52tr or cRNA. Cells transfected with the pGFP-PAC replicon and either subjected to a second mock-transfection or treated with 1 µg cycloheximide (cyclo) µl −1 were included as controls. Cells were lysed 24 h post-treatment and firefly and Renilla luciferase units (RLU) were measured using the dual-luciferase reporter assay system (Promega). A construct (pRF) containing only a cap-mediated Renilla luciferase reporter was included as a further control. Data are means± se of three experiments. Statistical analysis was conducted using a t- test ( P

    Journal: The Journal of General Virology

    Article Title: Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

    doi: 10.1099/vir.0.067751-0

    Figure Lengend Snippet: Effect of aptamers on translation. BHK-21 cells were transfected with a construct containing Renilla and firefly luciferase reporter genes under either (a) cap- or (b) FMDV IRES-mediated translation respectively (pRFMDVF). At 24 h post-transfection of the constructs, cells were subjected to a second transfection with aptamers 2′F-Cy3-47tr, 2′F-Cy3-52tr or cRNA. Cells transfected with the pGFP-PAC replicon and either subjected to a second mock-transfection or treated with 1 µg cycloheximide (cyclo) µl −1 were included as controls. Cells were lysed 24 h post-treatment and firefly and Renilla luciferase units (RLU) were measured using the dual-luciferase reporter assay system (Promega). A construct (pRF) containing only a cap-mediated Renilla luciferase reporter was included as a further control. Data are means± se of three experiments. Statistical analysis was conducted using a t- test ( P

    Article Snippet: Complete Dulbecco’s modified Eagle’s medium (DMEM) was removed from the BHK-21 cells and replaced with the ESCORT reagent mixture (Sigma-Aldrich).

    Techniques: Transfection, Construct, Luciferase, Reporter Assay

    Effect of aptamers on 3D pol expression in BHK-21 cells transfected with pGFP-PAC replicon. Cell extracts were prepared at 4 h post-transfection. (a) Aliquots were analysed by SDS-PAGE and immunoblotting for the presence of FMDV 3D pol and cellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells transfected with a Δ3D pol replicon (pGFP-PAC-Δ3D), mock-transfected or transfected with cRNA were included as controls. (b) Densitometry was conducted using ImageJ imaging analysis software on triplicate experiments for 3D pol expression, normalized to GAPDH. Data show mean values with sd ( n = 3) and statistical analysis performed using two-tailed paired t -test (* = P

    Journal: The Journal of General Virology

    Article Title: Inhibition of the foot-and-mouth disease virus subgenomic replicon by RNA aptamers

    doi: 10.1099/vir.0.067751-0

    Figure Lengend Snippet: Effect of aptamers on 3D pol expression in BHK-21 cells transfected with pGFP-PAC replicon. Cell extracts were prepared at 4 h post-transfection. (a) Aliquots were analysed by SDS-PAGE and immunoblotting for the presence of FMDV 3D pol and cellular glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Cells transfected with a Δ3D pol replicon (pGFP-PAC-Δ3D), mock-transfected or transfected with cRNA were included as controls. (b) Densitometry was conducted using ImageJ imaging analysis software on triplicate experiments for 3D pol expression, normalized to GAPDH. Data show mean values with sd ( n = 3) and statistical analysis performed using two-tailed paired t -test (* = P

    Article Snippet: Complete Dulbecco’s modified Eagle’s medium (DMEM) was removed from the BHK-21 cells and replaced with the ESCORT reagent mixture (Sigma-Aldrich).

    Techniques: Expressing, Transfection, SDS Page, Imaging, Software, Two Tailed Test

    In vitro ). TR, terminal repeats. (b, c) Detection of viral proteins in infected BHK-21 (3 PFU/cell, 6 h) cellular lysates using Western blotting (WB). kLANA was detected with MAb LN53, and mLANA was detected with MAb 6A3. Blotting against the MuHV-4 M3 protein and actin was used to assess levels of infection and loading, respectively. In panel b, expression of vCyclin and YFP was confirmed with anti-vCyclin and anti-GFP immunoblotting, respectively. (d) Images (confocal slices) depicting localization of LANA proteins in BHK-21 cells infected as described for panel a. DNA was stained with DAPI. Bar, 10 μm. (e) Relative levels (ΔΔ C T ) of M11, M3, and ORF63 in infected BHK-21 cells (5 PFU/cell, 8 h) compared to uninfected cells, normalized to GAPDH. Bars indicate mean fold changes ± standard deviations (SD). (f) Growth curves in BHK-21 cells infected with 0.01 PFU/cell. Total virus titers were determined. Time zero indicates input of virus after washing inoculum. Virus titers did not differ significantly between infection groups (Kruskal-Wallis test).

    Journal: Journal of Virology

    Article Title: In Vivo Persistence of Chimeric Virus after Substitution of the Kaposi's Sarcoma-Associated Herpesvirus LANA DNA Binding Domain with That of Murid Herpesvirus 4

    doi: 10.1128/JVI.01251-18

    Figure Lengend Snippet: In vitro ). TR, terminal repeats. (b, c) Detection of viral proteins in infected BHK-21 (3 PFU/cell, 6 h) cellular lysates using Western blotting (WB). kLANA was detected with MAb LN53, and mLANA was detected with MAb 6A3. Blotting against the MuHV-4 M3 protein and actin was used to assess levels of infection and loading, respectively. In panel b, expression of vCyclin and YFP was confirmed with anti-vCyclin and anti-GFP immunoblotting, respectively. (d) Images (confocal slices) depicting localization of LANA proteins in BHK-21 cells infected as described for panel a. DNA was stained with DAPI. Bar, 10 μm. (e) Relative levels (ΔΔ C T ) of M11, M3, and ORF63 in infected BHK-21 cells (5 PFU/cell, 8 h) compared to uninfected cells, normalized to GAPDH. Bars indicate mean fold changes ± standard deviations (SD). (f) Growth curves in BHK-21 cells infected with 0.01 PFU/cell. Total virus titers were determined. Time zero indicates input of virus after washing inoculum. Virus titers did not differ significantly between infection groups (Kruskal-Wallis test).

    Article Snippet: Viruses were reconstituted by transfection of recombinant BAC DNA into BHK-21 cells using X-tremeGENE HP (Roche).

    Techniques: In Vitro, Infection, Western Blot, Expressing, Staining

    Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

    Journal: Journal of Virology

    Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

    doi: 10.1128/JVI.79.18.11813-11823.2005

    Figure Lengend Snippet: Immunofluorescence staining of protein E in BHK-21 cells at 37°C (A) and 28°C (B). Cells were transfected with in vitro RNA transcripts of the clones indicated on the left (left panels) and stained with a polyclonal serum detecting protein

    Article Snippet: BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

    Techniques: Immunofluorescence, Staining, Transfection, In Vitro, Clone Assay

    Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

    Journal: Journal of Virology

    Article Title: Resuscitating Mutations in a Furin Cleavage-Deficient Mutant of the Flavivirus Tick-Borne Encephalitis Virus

    doi: 10.1128/JVI.79.18.11813-11823.2005

    Figure Lengend Snippet: Release of protein E from BHK-21 cells at 37°C (filled circles) and 28°C (empty circles). Cells were transfected with the mutant RNA prM(ΔR88), prM(ΔR88/C79Y), or prM(ΔR88/Y76C), as indicated. The release of protein

    Article Snippet: BHK-21 cells were grown in Eagle's minimal essential medium supplemented with 5% fetal calf serum, 1% glutamine, and 0.5% neomycin at 37°C (or, in some experiments, 28°C) in 5% CO2 .

    Techniques: Transfection, Mutagenesis

    Effect of blocking de novo synthesis of PI(4,5)P 2 with 1-butanol on FMDV and VSV internalization. (A) BHK-21 cells transfected (24 h) with PH-PLC-eGFP (green) were treated or not with 1.5% 1-butanol or 2-butanol for 5 min and then fixed and observed by confocal microscopy. Nuclei were stained using ToPro-3 (blue). Bar 10 µm. (B) Treatment with 1-butanol inhibits clathrin-dependent endocytosis. BHK-21 cells were treated as in (A) were incubated with fluorescent TF and processed as described in the legend of Fig. 1 . Bar: 10 µm. (C) Reduction of the ability of cells to internalize FMDV and VSV upon 1-butanol treatment. Cells treated as in (A) were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min in the presence of the drugs. Bars represent the mean percentage of cells with internalized virions ± SD, normalized to the level of cells with internalized virions in control samples. At least 500 cells per coverslip were scored for each case (3 coverslips). Asterisks denote statistically significant differences (ANOVA P≤0.05).

    Journal: PLoS ONE

    Article Title: Plasma Membrane Phosphatidylinositol 4,5 Bisphosphate Is Required for Internalization of Foot-and-Mouth Disease Virus and Vesicular Stomatitis Virus

    doi: 10.1371/journal.pone.0045172

    Figure Lengend Snippet: Effect of blocking de novo synthesis of PI(4,5)P 2 with 1-butanol on FMDV and VSV internalization. (A) BHK-21 cells transfected (24 h) with PH-PLC-eGFP (green) were treated or not with 1.5% 1-butanol or 2-butanol for 5 min and then fixed and observed by confocal microscopy. Nuclei were stained using ToPro-3 (blue). Bar 10 µm. (B) Treatment with 1-butanol inhibits clathrin-dependent endocytosis. BHK-21 cells were treated as in (A) were incubated with fluorescent TF and processed as described in the legend of Fig. 1 . Bar: 10 µm. (C) Reduction of the ability of cells to internalize FMDV and VSV upon 1-butanol treatment. Cells treated as in (A) were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min in the presence of the drugs. Bars represent the mean percentage of cells with internalized virions ± SD, normalized to the level of cells with internalized virions in control samples. At least 500 cells per coverslip were scored for each case (3 coverslips). Asterisks denote statistically significant differences (ANOVA P≤0.05).

    Article Snippet: BHK-21 cells were transfected using Lipofectamine Plus (Invitrogen) as described by the manufacturer or electroparated with the corresponding plasmid using Gene Pulser XCell™ (Bio Rad).

    Techniques: Blocking Assay, Transfection, Planar Chromatography, Confocal Microscopy, Staining, Incubation

    Depletion of PI(4,5)P 2 from plasma membrane after rapamycin-induced membrane targeting of an inositol 5-phosphatase. BHK-21 cells were cotransfected with PM-FRB-CFP (blue), mRFP-FKBP-dom5ptase (red) and PH-PLC-eGFP (green) plasmids using Lipofectamine Plus. At 24 h post-transfection, cells were treated with 10 nM rapamycin (10 min) to induce the depletion of PI(4,5)P 2 from plasma membrane. Then cells were fixed and observed by confocal microscopy. A representative example of a co-transfected cell is shown. See text for details regarding the inducible system for PI(4,5)P 2 depletion. Differential interference contrast (DIC) images are also shown. Bar: 10 µm.

    Journal: PLoS ONE

    Article Title: Plasma Membrane Phosphatidylinositol 4,5 Bisphosphate Is Required for Internalization of Foot-and-Mouth Disease Virus and Vesicular Stomatitis Virus

    doi: 10.1371/journal.pone.0045172

    Figure Lengend Snippet: Depletion of PI(4,5)P 2 from plasma membrane after rapamycin-induced membrane targeting of an inositol 5-phosphatase. BHK-21 cells were cotransfected with PM-FRB-CFP (blue), mRFP-FKBP-dom5ptase (red) and PH-PLC-eGFP (green) plasmids using Lipofectamine Plus. At 24 h post-transfection, cells were treated with 10 nM rapamycin (10 min) to induce the depletion of PI(4,5)P 2 from plasma membrane. Then cells were fixed and observed by confocal microscopy. A representative example of a co-transfected cell is shown. See text for details regarding the inducible system for PI(4,5)P 2 depletion. Differential interference contrast (DIC) images are also shown. Bar: 10 µm.

    Article Snippet: BHK-21 cells were transfected using Lipofectamine Plus (Invitrogen) as described by the manufacturer or electroparated with the corresponding plasmid using Gene Pulser XCell™ (Bio Rad).

    Techniques: Planar Chromatography, Transfection, Confocal Microscopy

    PIP5K-Iα is involved on entry and infection of FMDV C-S8c1 and VSV. (A) BHK-21 cells transfected with mCherry fused to WT or a KD version of PIP5K-Iα (mCherry-PIP5K-Iα WT and mCherry-PIP5K-Iα D268A, respectively) and 24 h later were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min and processed for immunofluorescence. The graph represents the percentage of cells that showed internalized virus determined as described in Materials and Methods . At least 100 transfected cells per coverslip were scored for each assay (3 coverslip). (B) BHK-21 cells were electroporated with a plasmid encoding mCherry-PIP5K-Iα WT as control, or mCherry-PIP5K-Iα D268A. At 24 h post-electroporation, monolayers were infected with the corresponding virus (MOI of 1 PFU/cell) and cells were fixed and processed for immunofluorescence at 7 h post-infection. Bars represent the mean percentage of transfected and infected cells ± SD, normalized to the level of infection of cells expressing the mCherry-PIP5K-Iα WT. Statistically significant differences between cells transfected with mCherry-PIP5K-Iα WT or D268A are indicated by an asterisk (ANOVA P≤0.05).

    Journal: PLoS ONE

    Article Title: Plasma Membrane Phosphatidylinositol 4,5 Bisphosphate Is Required for Internalization of Foot-and-Mouth Disease Virus and Vesicular Stomatitis Virus

    doi: 10.1371/journal.pone.0045172

    Figure Lengend Snippet: PIP5K-Iα is involved on entry and infection of FMDV C-S8c1 and VSV. (A) BHK-21 cells transfected with mCherry fused to WT or a KD version of PIP5K-Iα (mCherry-PIP5K-Iα WT and mCherry-PIP5K-Iα D268A, respectively) and 24 h later were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min and processed for immunofluorescence. The graph represents the percentage of cells that showed internalized virus determined as described in Materials and Methods . At least 100 transfected cells per coverslip were scored for each assay (3 coverslip). (B) BHK-21 cells were electroporated with a plasmid encoding mCherry-PIP5K-Iα WT as control, or mCherry-PIP5K-Iα D268A. At 24 h post-electroporation, monolayers were infected with the corresponding virus (MOI of 1 PFU/cell) and cells were fixed and processed for immunofluorescence at 7 h post-infection. Bars represent the mean percentage of transfected and infected cells ± SD, normalized to the level of infection of cells expressing the mCherry-PIP5K-Iα WT. Statistically significant differences between cells transfected with mCherry-PIP5K-Iα WT or D268A are indicated by an asterisk (ANOVA P≤0.05).

    Article Snippet: BHK-21 cells were transfected using Lipofectamine Plus (Invitrogen) as described by the manufacturer or electroparated with the corresponding plasmid using Gene Pulser XCell™ (Bio Rad).

    Techniques: Infection, Transfection, Incubation, Immunofluorescence, Plasmid Preparation, Electroporation, Expressing

    Functional requirement for dynamin of FMDV and VSV infection. (A) BHK-21 cells transfected with eGFP fused to WT or a DN version of dynamin (eGFP-Dyn WT and eGFP-Dyn K44A, respectively) and 24 h later were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min and processed for immunofluorescence. Nuclei were stained using ToPro-3 (blue). GFP and viruses are shown in green and red, respectively. Bar: 10 µm. (B) BHK-21 cells transfected and infected as in (A). The graph represents the percentage of cells that showed internalized virus, determined as described in Materials and Methods . At least 100 transfected cells per coverslip were scored in each assay (3 coverslip). (C) BHK-21 cells were electroporated with a plasmid encoding eGFP-Dyn WT as control, or eGFP-Dyn K44A. At 24 h post-electroporation, monolayers were infected with the corresponding virus (MOI of 1 PFU/cell). Cells were fixed and processed for immunofluorescence at 7 h post-infection. Bars represent the mean percentage of transfected and infected cells ± SD, normalized to the level of infection of cells expressing the eGFP-Dyn WT. Statistically significant differences between cells transfected with eGFP-Dyn WT or K44A are indicated by an asterisk (ANOVA P≤0.05).

    Journal: PLoS ONE

    Article Title: Plasma Membrane Phosphatidylinositol 4,5 Bisphosphate Is Required for Internalization of Foot-and-Mouth Disease Virus and Vesicular Stomatitis Virus

    doi: 10.1371/journal.pone.0045172

    Figure Lengend Snippet: Functional requirement for dynamin of FMDV and VSV infection. (A) BHK-21 cells transfected with eGFP fused to WT or a DN version of dynamin (eGFP-Dyn WT and eGFP-Dyn K44A, respectively) and 24 h later were incubated with the different FMDV variants (C-S8c1 and MARLS) or VSV (MOI of 70 PFU/cell) for 25 min and processed for immunofluorescence. Nuclei were stained using ToPro-3 (blue). GFP and viruses are shown in green and red, respectively. Bar: 10 µm. (B) BHK-21 cells transfected and infected as in (A). The graph represents the percentage of cells that showed internalized virus, determined as described in Materials and Methods . At least 100 transfected cells per coverslip were scored in each assay (3 coverslip). (C) BHK-21 cells were electroporated with a plasmid encoding eGFP-Dyn WT as control, or eGFP-Dyn K44A. At 24 h post-electroporation, monolayers were infected with the corresponding virus (MOI of 1 PFU/cell). Cells were fixed and processed for immunofluorescence at 7 h post-infection. Bars represent the mean percentage of transfected and infected cells ± SD, normalized to the level of infection of cells expressing the eGFP-Dyn WT. Statistically significant differences between cells transfected with eGFP-Dyn WT or K44A are indicated by an asterisk (ANOVA P≤0.05).

    Article Snippet: BHK-21 cells were transfected using Lipofectamine Plus (Invitrogen) as described by the manufacturer or electroparated with the corresponding plasmid using Gene Pulser XCell™ (Bio Rad).

    Techniques: Functional Assay, Infection, Transfection, Incubation, Immunofluorescence, Staining, Plasmid Preparation, Electroporation, Expressing

    Effect of PI(4,5)P 2 depletion by ionomycin on FMDV and VSV internalization. (A) Visualization of PI(4,5)P 2 depletion from plasma membrane. BHK-21 cells transfected (24 h) with PH-PLC-eGFP, encoding a reporter protein for PI(4,5)P 2 fused to GFP (green), were treated or not with 5 µM ionomycin 30 min and then fixed and observed by confocal microscopy. Nuclei were stained using ToPro-3 (blue). Bar 10 µm. (B) Treatment with ionomycin inhibits clathrin-mediated endocytosis. BHK-21 cells, treated with ionomycin as in (A), were incubated with Alexa Fluor 488-labelled TF (green) for 5 min in the presence of the drug and extracellular TF was eliminated by acid wash as described [32] . Cells were fixed and nuclei were stained using DAPI (blue). Bar: 10 µm. (C) Inhibition of the ability of cells to internalize FMDV and VSV upon ionomycin treatment. Cells treated with ionomycin as in (A) were incubated with the different FMDV variants (C-S8c1 and MARLS) or with VSV (MOI of 70 PFU/cell) for 25 min in the presence of ionomycin. Cells were fixed and processed for immunofluorescence to stain viral particles as described in Materials and Methods . Bars represent the mean percentage of cells with internalized virions ± SD, normalized to the level of cells with internalized virions in control samples. At least 500 cells per coverslip were scored for each case (3 coverslips). Asterisks denote statistically significant differences (ANOVA P≤0.05).

    Journal: PLoS ONE

    Article Title: Plasma Membrane Phosphatidylinositol 4,5 Bisphosphate Is Required for Internalization of Foot-and-Mouth Disease Virus and Vesicular Stomatitis Virus

    doi: 10.1371/journal.pone.0045172

    Figure Lengend Snippet: Effect of PI(4,5)P 2 depletion by ionomycin on FMDV and VSV internalization. (A) Visualization of PI(4,5)P 2 depletion from plasma membrane. BHK-21 cells transfected (24 h) with PH-PLC-eGFP, encoding a reporter protein for PI(4,5)P 2 fused to GFP (green), were treated or not with 5 µM ionomycin 30 min and then fixed and observed by confocal microscopy. Nuclei were stained using ToPro-3 (blue). Bar 10 µm. (B) Treatment with ionomycin inhibits clathrin-mediated endocytosis. BHK-21 cells, treated with ionomycin as in (A), were incubated with Alexa Fluor 488-labelled TF (green) for 5 min in the presence of the drug and extracellular TF was eliminated by acid wash as described [32] . Cells were fixed and nuclei were stained using DAPI (blue). Bar: 10 µm. (C) Inhibition of the ability of cells to internalize FMDV and VSV upon ionomycin treatment. Cells treated with ionomycin as in (A) were incubated with the different FMDV variants (C-S8c1 and MARLS) or with VSV (MOI of 70 PFU/cell) for 25 min in the presence of ionomycin. Cells were fixed and processed for immunofluorescence to stain viral particles as described in Materials and Methods . Bars represent the mean percentage of cells with internalized virions ± SD, normalized to the level of cells with internalized virions in control samples. At least 500 cells per coverslip were scored for each case (3 coverslips). Asterisks denote statistically significant differences (ANOVA P≤0.05).

    Article Snippet: BHK-21 cells were transfected using Lipofectamine Plus (Invitrogen) as described by the manufacturer or electroparated with the corresponding plasmid using Gene Pulser XCell™ (Bio Rad).

    Techniques: Transfection, Planar Chromatography, Confocal Microscopy, Staining, Incubation, Inhibition, Immunofluorescence

    Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. BHK-21 cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.

    Journal: Genome Biology and Evolution

    Article Title: Nonreplicative RNA Recombination of an Animal Plus-Strand RNA Virus in the Absence of Efficient Translation of Viral Proteins

    doi: 10.1093/gbe/evx046

    Figure Lengend Snippet: Processing of fusion proteins of recombinants R-1, R-2, and R-3. ( A ) Schematic representation of authentic (P-1, P-2, P-3) and mutated (P-2P, P-3P) fusion proteins transiently expressed in the MVA-T7 system. P-1 encompasses the wt proteins N pro and C, P-2 and P-3 comprise N pro and the C fusion proteins encoded by the recombinant viruses R-2 and R-3, respectively. In P-2P and P-3P, the asterisks mark the serine to proline substitutions directly downstream of the ubiquitin fragments. Black arrows indicate autoproteolytic cleavage mediated by N pro . White arrows (P-2 and P-3) indicate partial processing directly downstream of the ubiquitin fragments. ( B and C ) Western blot analysis. BHK-21 cells were infected with MVA-T7 and transfected with pCITE-N pro -C constructs encoding P-1, P-2, P-2P, P-3, and P-3P, respectively. Nontransfected cells (−) served as negative control. Cells were lysed 20 h posttransfection and analyzed by Western blot using MAb 1F7 (panel B ) and MAb 13B6 (panel C ) to detect C (C-ubi-C) and N pro , respectively.

    Article Snippet: Baby hamster kidney (BHK-21) cells were obtained from the DSMZ (Braunschweig, Germany) and maintained in EDulb medium supplemented with 5% fetal calf serum.

    Techniques: Recombinant, Western Blot, Infection, Transfection, Construct, Negative Control

    Testing the interaction between Mtk and SdhB in BHK-21 cells. The interaction between Mtk and SdhB was tested using the F2H assay 24 h after the transfection of BHK-21 cells. ( a ) DAPI channel. ( b ) GFP channel. ( c ) RFP channel. ( d ) Merged. The presence of both green and red spots (arrows) in the GFP and RFP channels, respectively, shows the interaction between Mtk and SdhB as confirmed by the presence of both green and red spots. Scale bars = 25 µm.

    Journal: Scientific Reports

    Article Title: The selective antifungal activity of Drosophila melanogaster metchnikowin reflects the species-dependent inhibition of succinate–coenzyme Q reductase

    doi: 10.1038/s41598-017-08407-x

    Figure Lengend Snippet: Testing the interaction between Mtk and SdhB in BHK-21 cells. The interaction between Mtk and SdhB was tested using the F2H assay 24 h after the transfection of BHK-21 cells. ( a ) DAPI channel. ( b ) GFP channel. ( c ) RFP channel. ( d ) Merged. The presence of both green and red spots (arrows) in the GFP and RFP channels, respectively, shows the interaction between Mtk and SdhB as confirmed by the presence of both green and red spots. Scale bars = 25 µm.

    Article Snippet: Genetically modified BHK-21 cells (F2H® cells, BioCat) were co-transfected with the pTagGFP2 and pTagRFP constructs.

    Techniques: Transfection

    S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on BHK-21 cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.

    Journal: Journal of Virology

    Article Title: Sindbis-Group Alphavirus Replication in Periosteum and Endosteum of Long Bones in Adult Mice

    doi:

    Figure Lengend Snippet: S.A.AR86, as well as other Sindbis-group alphaviruses, replicates in bone- and joint-associated tissues. (A) Six-week-old female CD-1 mice were infected with 10 3 PFU of s55 i.v. Three days postinfection mice were sacrificed, exsanguinated, and perfused with PBS, and their femurs and quadriceps muscles were removed by dissection and titrated for infectious virus on BHK-21 cells. Data are shown as log PFU per gram of tissue for femur and muscle and as PFU per milliliter for serum. Each bar represents a single animal. The arrow indicates the limit of detection, and asterisks denote samples below the limit of detection. Data shown are from one of four experiments. (B) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of s51 and sacrificed 3 days postinfection, and right femurs were removed for virus titration. Bone marrow was aspirated from the diaphyses of the femurs using 0.4 ml of PBS–1% donor calf serum per femur. Aspirates were freeze-thawed and titrated for infectious virus by plaque assay. Following marrow aspiration, the remaining femoral tissue was processed as for panel A and titrated for infectious virus by plaque assay. Titer is shown as total PFU per marrow aspirate (Asp) or femur (without aspirated marrow), with each bar representing a single animal. The arrow indicates the limit of detection, and asterisks denote samples with titers below the limit of detection. Shown is one of three comparable experiments. (C) Six-week-old female CD-1 mice were infected i.v. with 10 3 PFU of the virus s55, TR339, or TRSB. Three days postinfection mice were sacrificed and both femurs were removed for virus titration. Femurs were processed and titrated for infectious virus as for panel A. Each bar represents results from a single animal. The arrow indicates the limit of detection. N/D, not done. Shown is one of two comparable experiments.

    Article Snippet: The tissue homogenate was then clarified by centrifugation and assayed for infectious virus by plaque assay on BHK-21 cells (ATCC CRL 8544) as previously described ( ).

    Techniques: Mouse Assay, Infection, Dissection, Titration, Plaque Assay

    Detection of the FMDV structure protein and FMDV capsid. Immunofluoresence was used to determine the expression levels of the FMDV structure protein following transfection with either pcDNA3.1(+) (a), pA (b) pB (c) or pC (d). FMDV capsid was observed by TEM of BHK-21 cells transfected with pA (e) or pC (d).

    Journal: PLoS ONE

    Article Title: Intranasal Delivery of Cationic PLGA Nano/Microparticles- Loaded FMDV DNA Vaccine Encoding IL-6 Elicited Protective Immunity against FMDV Challenge

    doi: 10.1371/journal.pone.0027605

    Figure Lengend Snippet: Detection of the FMDV structure protein and FMDV capsid. Immunofluoresence was used to determine the expression levels of the FMDV structure protein following transfection with either pcDNA3.1(+) (a), pA (b) pB (c) or pC (d). FMDV capsid was observed by TEM of BHK-21 cells transfected with pA (e) or pC (d).

    Article Snippet: Transfections Baby hamster kidney (BHK-21) cells (Boster, Wuhan, China) (4×105 ) were seeded onto cover slips on six-well plates and incubated at 37°C in a CO2 incubator until the cells were 80% confluent.

    Techniques: Expressing, Transfection, Transmission Electron Microscopy

    Characterization of FMDV protein and IL-6 production in BHK-21 cells. (A)Two days after transfection with either pA, pB or pC, and negative control (PBS) and irrelevant control (Bovine Serum Albumin, BSA) were added; BHK-21 cells were analyzed for expression of FMDV proteins by sandwich-ELISA. BHK-21 cell lysates were diluted twofold. The data are expressed as the mean OD for each dilution. (B) Expression of IL-6 was determined by assessing IL-6 levels in BHK-21 cell lysates by ELISA two-days after transfection. The data are expressed as the mean OD ± SEM, measured in duplicate. Means were compared by non-parametric ANOVA.

    Journal: PLoS ONE

    Article Title: Intranasal Delivery of Cationic PLGA Nano/Microparticles- Loaded FMDV DNA Vaccine Encoding IL-6 Elicited Protective Immunity against FMDV Challenge

    doi: 10.1371/journal.pone.0027605

    Figure Lengend Snippet: Characterization of FMDV protein and IL-6 production in BHK-21 cells. (A)Two days after transfection with either pA, pB or pC, and negative control (PBS) and irrelevant control (Bovine Serum Albumin, BSA) were added; BHK-21 cells were analyzed for expression of FMDV proteins by sandwich-ELISA. BHK-21 cell lysates were diluted twofold. The data are expressed as the mean OD for each dilution. (B) Expression of IL-6 was determined by assessing IL-6 levels in BHK-21 cell lysates by ELISA two-days after transfection. The data are expressed as the mean OD ± SEM, measured in duplicate. Means were compared by non-parametric ANOVA.

    Article Snippet: Transfections Baby hamster kidney (BHK-21) cells (Boster, Wuhan, China) (4×105 ) were seeded onto cover slips on six-well plates and incubated at 37°C in a CO2 incubator until the cells were 80% confluent.

    Techniques: Transfection, Negative Control, Expressing, Sandwich ELISA, Enzyme-linked Immunosorbent Assay