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Millipore rosetta bl21 de3 e coli cells
Rosetta Bl21 De3 E Coli Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore competent bl21 de3 e coli cells
Competent Bl21 De3 E Coli Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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competent bl21 de3 e coli cells - by Bioz Stars, 2023-01
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Millipore e coli bl21 de3 cells
( A ) IbeA+ <t>E.</t> <t>coli</t> K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).
E Coli Bl21 De3 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/e coli bl21 de3 cells/product/Millipore
Average 86 stars, based on 1 article reviews
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e coli bl21 de3 cells - by Bioz Stars, 2023-01
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1) Product Images from "Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells"

Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0035862

( A ) IbeA+ E. coli K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).
Figure Legend Snippet: ( A ) IbeA+ E. coli K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).

Techniques Used: Activation Assay, Incubation, Transmigration Assay, Positive Control

( A ) HBMECs were incubated with or without PD098059 (50 µM) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). ( B ) HBMECs were incubated with or without ERK89 (vimentin-binding domain, 25 µg/ml) and ERK312 (control peptide, 25 µg/ml) for 60 min before infection with E44 or ZD1 (10 7 /ml). In both ( A ) and ( B ), ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β) and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of infection with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.
Figure Legend Snippet: ( A ) HBMECs were incubated with or without PD098059 (50 µM) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). ( B ) HBMECs were incubated with or without ERK89 (vimentin-binding domain, 25 µg/ml) and ERK312 (control peptide, 25 µg/ml) for 60 min before infection with E44 or ZD1 (10 7 /ml). In both ( A ) and ( B ), ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β) and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of infection with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

Techniques Used: Incubation, Binding Assay, Infection, Translocation Assay

( A ) Immunofluorescence microscopy was used to examine the correlation between vimentin reorganization and NF-κB translocation to the nucleus after 2 h of stimulation with IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization of vimentin and NF-κB (p65) Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced NF-κB activation in HBMECs by siRNA-mediated knockdown of vimentin. HBMECs were transfected with vimentin or control siRNA as described in . After 24 h incubation, the cells were treated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. Vimentin (VIM), α7 nAChR, ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, and PSF re-localization were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; VIM KD: HBMECs transfected with vimentin siRNA; UNT: Untreated HBMECs.
Figure Legend Snippet: ( A ) Immunofluorescence microscopy was used to examine the correlation between vimentin reorganization and NF-κB translocation to the nucleus after 2 h of stimulation with IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization of vimentin and NF-κB (p65) Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced NF-κB activation in HBMECs by siRNA-mediated knockdown of vimentin. HBMECs were transfected with vimentin or control siRNA as described in . After 24 h incubation, the cells were treated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. Vimentin (VIM), α7 nAChR, ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, and PSF re-localization were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; VIM KD: HBMECs transfected with vimentin siRNA; UNT: Untreated HBMECs.

Techniques Used: Immunofluorescence, Microscopy, Translocation Assay, Staining, Activation Assay, Transfection, Incubation

( A ) IbeA− and IbeA+ E. coli K1-induced β-tubulin/vimentin clustering and colocalization. Immunofluorescence microscopy was used to examine the clustering and reorganization of vimentin and β-tubulin after 2 h of incubation with the IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against β-tubulin conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization between vimentin and β-tubulin around the perinuclear region. Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced cytoplasmic activation and nuclear translocation of NF-κB (p65) in HBMECs by the microtubule inhibitors. HBMECs were incubated with or without colchicines (Col, 2 µM), vincristine (Vin, 1 µM), nocodazole (Noc, 25 µg/ml) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). Phosphorylation of ERK1/2 (p-Erk1/2) and IKK α/β (p-IKK α/β) was examined in cytoplasmic fractions after 30 min of E. coli K1 treatment. NF-κB (p65) translocation to nucleus in nuclear fractions was examined after 2 h of E. coli K1 incubation. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.
Figure Legend Snippet: ( A ) IbeA− and IbeA+ E. coli K1-induced β-tubulin/vimentin clustering and colocalization. Immunofluorescence microscopy was used to examine the clustering and reorganization of vimentin and β-tubulin after 2 h of incubation with the IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against β-tubulin conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization between vimentin and β-tubulin around the perinuclear region. Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced cytoplasmic activation and nuclear translocation of NF-κB (p65) in HBMECs by the microtubule inhibitors. HBMECs were incubated with or without colchicines (Col, 2 µM), vincristine (Vin, 1 µM), nocodazole (Noc, 25 µg/ml) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). Phosphorylation of ERK1/2 (p-Erk1/2) and IKK α/β (p-IKK α/β) was examined in cytoplasmic fractions after 30 min of E. coli K1 treatment. NF-κB (p65) translocation to nucleus in nuclear fractions was examined after 2 h of E. coli K1 incubation. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

Techniques Used: Immunofluorescence, Microscopy, Incubation, Staining, Activation Assay, Translocation Assay

HBMECs were transfected with PSF or control siRNA as described in . IbeA+ E. coli K1 penetration ( A ) and PMN transmigration ( B ) across siRNA-transfected HBMECs were performed as described in the . Both invasion and PMN transmigration assays were performed in triplicates. Results for invasion are expressed as a relative percentage compared to the penetration rate of E44 in the siRNA control (CON) (100%). Results for PMN transmigration are expressed as the percentage of PMN transmigration of total PMNs. The control siRNA-transfected HBMECs infected with E44 and ZD1 were used as the controls (panels A and B). The significant differences regarding to the control were marked by asterisks (*P<0.05; **P<0.01). ( C ) After transfection, the cells were stimulated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. PSF re-localization, p-Erk1/2, p-IKK α/β and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) and PSF in nuclear fractions were examined after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; PSF KD, HBMECs transfected with PSF siRNA; UNT: Untreated HBMECs. ( D ) Time course analysis of IbeA-induced tyrosine phosphorylation of PSF. HBMECs were incubated with the IbeA protein (0.1 µg/ml) for 2, 6, and 24 hrs, respectively. The cytoplasmic fractions were extracted and immunoprecipitated (IP) using the anti-phosphotyrosine antibody as described in . The Tyr-IP complexes were subjected to Western blot using the mouse monoclonal antibody against PSF. Total mouse IgG was detected as an internal loading control. CON: the IP control without primary antibody incubation; 0 h: the control HBMECs without IbeA incubation.
Figure Legend Snippet: HBMECs were transfected with PSF or control siRNA as described in . IbeA+ E. coli K1 penetration ( A ) and PMN transmigration ( B ) across siRNA-transfected HBMECs were performed as described in the . Both invasion and PMN transmigration assays were performed in triplicates. Results for invasion are expressed as a relative percentage compared to the penetration rate of E44 in the siRNA control (CON) (100%). Results for PMN transmigration are expressed as the percentage of PMN transmigration of total PMNs. The control siRNA-transfected HBMECs infected with E44 and ZD1 were used as the controls (panels A and B). The significant differences regarding to the control were marked by asterisks (*P<0.05; **P<0.01). ( C ) After transfection, the cells were stimulated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. PSF re-localization, p-Erk1/2, p-IKK α/β and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) and PSF in nuclear fractions were examined after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; PSF KD, HBMECs transfected with PSF siRNA; UNT: Untreated HBMECs. ( D ) Time course analysis of IbeA-induced tyrosine phosphorylation of PSF. HBMECs were incubated with the IbeA protein (0.1 µg/ml) for 2, 6, and 24 hrs, respectively. The cytoplasmic fractions were extracted and immunoprecipitated (IP) using the anti-phosphotyrosine antibody as described in . The Tyr-IP complexes were subjected to Western blot using the mouse monoclonal antibody against PSF. Total mouse IgG was detected as an internal loading control. CON: the IP control without primary antibody incubation; 0 h: the control HBMECs without IbeA incubation.

Techniques Used: Transfection, Transmigration Assay, Infection, Incubation, Immunoprecipitation, Western Blot

Immunoblotting analysis of polyubiquitinylated proteins (Ub-prs): ( A ) HBMECs with siRNA-mediated knockdown of vimentin; and ( B ) HBMECs transduced with the lentivirus constructs expressing GFP–VRT and GFP. In both (A) and (B), all the cells were incubated with or without E. coli K1 strains (E44 and ZD1, 10 7 /ml) for 2 h. The Ub-prs were detected in cytoplasmic fractions to determine the proteasomal degradation as described in . β-actin was used as an internal loading control. In all experiments, untreated HBMECs (UNT) were taken as controls.
Figure Legend Snippet: Immunoblotting analysis of polyubiquitinylated proteins (Ub-prs): ( A ) HBMECs with siRNA-mediated knockdown of vimentin; and ( B ) HBMECs transduced with the lentivirus constructs expressing GFP–VRT and GFP. In both (A) and (B), all the cells were incubated with or without E. coli K1 strains (E44 and ZD1, 10 7 /ml) for 2 h. The Ub-prs were detected in cytoplasmic fractions to determine the proteasomal degradation as described in . β-actin was used as an internal loading control. In all experiments, untreated HBMECs (UNT) were taken as controls.

Techniques Used: Western Blot, Transduction, Construct, Expressing, Incubation


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Millipore bl21 de3 e coli competent cells
Bl21 De3 E Coli Competent Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore e coli bl21 de3 plyss prsetc cells
Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of <t>BL21</t> <t>(DE3)</t> <t>pLysS</t> (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS <t>(pRSETC)</t> as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).
E Coli Bl21 De3 Plyss Prsetc Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Display of the Viral Epitopes on Lactococcus lactis : A Model for Food Grade Vaccine against EV71"

Article Title: Display of the Viral Epitopes on Lactococcus lactis : A Model for Food Grade Vaccine against EV71

Journal: Biotechnology Research International

doi: 10.1155/2013/431315

Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of BL21 (DE3) pLysS (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS (pRSETC) as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).
Figure Legend Snippet: Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of BL21 (DE3) pLysS (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS (pRSETC) as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).

Techniques Used: Western Blot, Recombinant, Negative Control

Detection of serum antibody response against VP1 1-67aa and VP1 35-100aa epitopes of EV71 in mice immunized with L. lactis displaying AcmA/VP1 35-100aa . Lane 1: BSA protein; lane 2: E. coli (pRSET) total proteins; lane 3: total proteins of L. lactis MG1363; lane 4: purified AcmA/VP1 1-67aa proteins; lane 5: purified AcmA/VP1 35-100aa protein; lane M: protein marker (Fermentas, Hanover, MD, USA). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa) and AcmA/VP1 35-100aa (~25 kDa).
Figure Legend Snippet: Detection of serum antibody response against VP1 1-67aa and VP1 35-100aa epitopes of EV71 in mice immunized with L. lactis displaying AcmA/VP1 35-100aa . Lane 1: BSA protein; lane 2: E. coli (pRSET) total proteins; lane 3: total proteins of L. lactis MG1363; lane 4: purified AcmA/VP1 1-67aa proteins; lane 5: purified AcmA/VP1 35-100aa protein; lane M: protein marker (Fermentas, Hanover, MD, USA). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa) and AcmA/VP1 35-100aa (~25 kDa).

Techniques Used: Purification, Marker, Recombinant


Structured Review

Millipore e coli bl21 de3 cells
Images generated with The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC with data files obtained through the Protein Data Bank Europe. (a) Image of human annexin V generated from the crystal structure . (b) Image of <t>Escherichia</t> <t>coli</t> PNP . c Mesh image of a model of PNP-AV hexamer.
E Coli Bl21 De3 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy"

Article Title: Purine Nucleoside Phosphorylase Targeted by Annexin V to Breast Cancer Vasculature for Enzyme Prodrug Therapy

Journal: PLoS ONE

doi: 10.1371/journal.pone.0076403

Images generated with The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC with data files obtained through the Protein Data Bank Europe. (a) Image of human annexin V generated from the crystal structure . (b) Image of Escherichia coli PNP . c Mesh image of a model of PNP-AV hexamer.
Figure Legend Snippet: Images generated with The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC with data files obtained through the Protein Data Bank Europe. (a) Image of human annexin V generated from the crystal structure . (b) Image of Escherichia coli PNP . c Mesh image of a model of PNP-AV hexamer.

Techniques Used: Generated


Structured Review

Millipore bl21 de3 plyss e coli cells
Bl21 De3 Plyss E Coli Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore cacl 2 competent e coli bl21 de3 cells
Cacl 2 Competent E Coli Bl21 De3 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Millipore cacl 2 competent e coli bl21 de3 cells
Cacl 2 Competent E Coli Bl21 De3 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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single colony transformed e coli bl21 de3 rosetta cells  (Millipore)

 
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    Millipore single colony transformed e coli bl21 de3 rosetta cells
    Single Colony Transformed E Coli Bl21 De3 Rosetta Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rosetta bl21 de3 e coli cells
    Rosetta Bl21 De3 E Coli Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore competent bl21 de3 e coli cells
    Competent Bl21 De3 E Coli Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/competent bl21 de3 e coli cells/product/Millipore
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    86
    Millipore e coli bl21 de3 cells
    ( A ) IbeA+ <t>E.</t> <t>coli</t> K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).
    E Coli Bl21 De3 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bl21 de3 e coli competent cells
    ( A ) IbeA+ <t>E.</t> <t>coli</t> K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).
    Bl21 De3 E Coli Competent Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e coli bl21 de3 plyss prsetc cells
    Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of <t>BL21</t> <t>(DE3)</t> <t>pLysS</t> (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS <t>(pRSETC)</t> as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).
    E Coli Bl21 De3 Plyss Prsetc Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore bl21 de3 plyss e coli cells
    Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of <t>BL21</t> <t>(DE3)</t> <t>pLysS</t> (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS <t>(pRSETC)</t> as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).
    Bl21 De3 Plyss E Coli Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cacl 2 competent e coli bl21 de3 cells
    Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of <t>BL21</t> <t>(DE3)</t> <t>pLysS</t> (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS <t>(pRSETC)</t> as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).
    Cacl 2 Competent E Coli Bl21 De3 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore single colony transformed e coli bl21 de3 rosetta cells
    Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of <t>BL21</t> <t>(DE3)</t> <t>pLysS</t> (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS <t>(pRSETC)</t> as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).
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    Image Search Results


    ( A ) IbeA+ E. coli K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).

    Journal: PLoS ONE

    Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

    doi: 10.1371/journal.pone.0035862

    Figure Lengend Snippet: ( A ) IbeA+ E. coli K1 induced NF-κB activation in HBMECs was suppressed by CAPE. HBMECs were incubated with or without the NF-κB inhibitor CAPE (25 µM) for 30 min before stimulation with E44 or ZD1 (10 7 /mL). IKK α/β phosphorylation (p-IKK α/β) in cytoplasmic fractions and NF-κB (p65) in nuclear fractions was examined after 2 h of stimulation with E. coli strains. The β-actin in both fractions was detected as internal loading controls. CON, control without E. coli stimulation. ( B–C ) Effects of CAPE (0–25 µM) on IbeA+ E. coli K1 penetration and PMN transmigration across HBMECs were examined. HBMECs were incubated with various concentrations of CAPE for 1 h before the invasion and PMN transmigration assays. ( B ). E. coli (10 7 CFU) were added to the HBMEC monolayers after CAPE treatment. Invasion assays were carried out as described in the . ( C ) The CAPE-pretreated HBMECs were stimulated with E. coli (10 6 CFU) in the lower chamber for 2 h and incubated with PMN (10 6 ) in the upper chamber at 37°C for another 4 h. All assays were performed in triplicates. Results for invasion are expressed as relative invasion compared to the positive control without drug treatment (100%). Results for PMNT are expressed as the percentage of leukocyte transmigration of the total added. Both the invasion and PMNT assays were done with E44 (black column) and ZD1 (white column). E. coli meningitis was induced in neonatal mice with or without CAPE treatment (n = 5) as described in . ( D ) Recruitment of PMN into the CSF; ( E ) Flux of albumin into the CNS; and ( F ) Levels of soluble NF-κB (p65) in CSF. The significant differences with regard to the controls without CAPE treatment were marked by asterisks (*P<0.05; **P<0.01).

    Article Snippet: Expression and purification of recombinant proteins in E. coli BL21 (DE3) cells were carried out according to the manufacturer's instructions (EMD Bioscience).

    Techniques: Activation Assay, Incubation, Transmigration Assay, Positive Control

    ( A ) HBMECs were incubated with or without PD098059 (50 µM) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). ( B ) HBMECs were incubated with or without ERK89 (vimentin-binding domain, 25 µg/ml) and ERK312 (control peptide, 25 µg/ml) for 60 min before infection with E44 or ZD1 (10 7 /ml). In both ( A ) and ( B ), ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β) and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of infection with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

    Journal: PLoS ONE

    Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

    doi: 10.1371/journal.pone.0035862

    Figure Lengend Snippet: ( A ) HBMECs were incubated with or without PD098059 (50 µM) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). ( B ) HBMECs were incubated with or without ERK89 (vimentin-binding domain, 25 µg/ml) and ERK312 (control peptide, 25 µg/ml) for 60 min before infection with E44 or ZD1 (10 7 /ml). In both ( A ) and ( B ), ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β) and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of infection with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

    Article Snippet: Expression and purification of recombinant proteins in E. coli BL21 (DE3) cells were carried out according to the manufacturer's instructions (EMD Bioscience).

    Techniques: Incubation, Binding Assay, Infection, Translocation Assay

    ( A ) Immunofluorescence microscopy was used to examine the correlation between vimentin reorganization and NF-κB translocation to the nucleus after 2 h of stimulation with IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization of vimentin and NF-κB (p65) Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced NF-κB activation in HBMECs by siRNA-mediated knockdown of vimentin. HBMECs were transfected with vimentin or control siRNA as described in . After 24 h incubation, the cells were treated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. Vimentin (VIM), α7 nAChR, ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, and PSF re-localization were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; VIM KD: HBMECs transfected with vimentin siRNA; UNT: Untreated HBMECs.

    Journal: PLoS ONE

    Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

    doi: 10.1371/journal.pone.0035862

    Figure Lengend Snippet: ( A ) Immunofluorescence microscopy was used to examine the correlation between vimentin reorganization and NF-κB translocation to the nucleus after 2 h of stimulation with IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against NF-κB (p65) conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization of vimentin and NF-κB (p65) Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced NF-κB activation in HBMECs by siRNA-mediated knockdown of vimentin. HBMECs were transfected with vimentin or control siRNA as described in . After 24 h incubation, the cells were treated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. Vimentin (VIM), α7 nAChR, ERK1/2 phosphorylation (p-Erk1/2), IKK α/β phosphorylation (p-IKK α/β), IκBα degradation, and PSF re-localization were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) translocation to the nucleus was examined in nuclear fractions after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; VIM KD: HBMECs transfected with vimentin siRNA; UNT: Untreated HBMECs.

    Article Snippet: Expression and purification of recombinant proteins in E. coli BL21 (DE3) cells were carried out according to the manufacturer's instructions (EMD Bioscience).

    Techniques: Immunofluorescence, Microscopy, Translocation Assay, Staining, Activation Assay, Transfection, Incubation

    ( A ) IbeA− and IbeA+ E. coli K1-induced β-tubulin/vimentin clustering and colocalization. Immunofluorescence microscopy was used to examine the clustering and reorganization of vimentin and β-tubulin after 2 h of incubation with the IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against β-tubulin conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization between vimentin and β-tubulin around the perinuclear region. Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced cytoplasmic activation and nuclear translocation of NF-κB (p65) in HBMECs by the microtubule inhibitors. HBMECs were incubated with or without colchicines (Col, 2 µM), vincristine (Vin, 1 µM), nocodazole (Noc, 25 µg/ml) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). Phosphorylation of ERK1/2 (p-Erk1/2) and IKK α/β (p-IKK α/β) was examined in cytoplasmic fractions after 30 min of E. coli K1 treatment. NF-κB (p65) translocation to nucleus in nuclear fractions was examined after 2 h of E. coli K1 incubation. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

    Journal: PLoS ONE

    Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

    doi: 10.1371/journal.pone.0035862

    Figure Lengend Snippet: ( A ) IbeA− and IbeA+ E. coli K1-induced β-tubulin/vimentin clustering and colocalization. Immunofluorescence microscopy was used to examine the clustering and reorganization of vimentin and β-tubulin after 2 h of incubation with the IbeA protein (0.1 µg/ml), E44 or ZD1 (25 MOI). HBMECs were triple-stained with the V9 antibody against vimentin conjugated to FITC (green), the rabbit antibody against β-tubulin conjugated to rhodamine (red), and DAPI (blue). The merged images are shown in the right-hand panels (Merge). Arrows indicated cells with colocalization between vimentin and β-tubulin around the perinuclear region. Scale bar, 50 µm. ( B ) Blockage of IbeA+ E. coli K1-induced cytoplasmic activation and nuclear translocation of NF-κB (p65) in HBMECs by the microtubule inhibitors. HBMECs were incubated with or without colchicines (Col, 2 µM), vincristine (Vin, 1 µM), nocodazole (Noc, 25 µg/ml) for 60 min before stimulation with E44 or ZD1 (10 7 /ml). Phosphorylation of ERK1/2 (p-Erk1/2) and IKK α/β (p-IKK α/β) was examined in cytoplasmic fractions after 30 min of E. coli K1 treatment. NF-κB (p65) translocation to nucleus in nuclear fractions was examined after 2 h of E. coli K1 incubation. β-actin in both fractions was detected as internal loading controls. CON, control without bacterial stimulation.

    Article Snippet: Expression and purification of recombinant proteins in E. coli BL21 (DE3) cells were carried out according to the manufacturer's instructions (EMD Bioscience).

    Techniques: Immunofluorescence, Microscopy, Incubation, Staining, Activation Assay, Translocation Assay

    HBMECs were transfected with PSF or control siRNA as described in . IbeA+ E. coli K1 penetration ( A ) and PMN transmigration ( B ) across siRNA-transfected HBMECs were performed as described in the . Both invasion and PMN transmigration assays were performed in triplicates. Results for invasion are expressed as a relative percentage compared to the penetration rate of E44 in the siRNA control (CON) (100%). Results for PMN transmigration are expressed as the percentage of PMN transmigration of total PMNs. The control siRNA-transfected HBMECs infected with E44 and ZD1 were used as the controls (panels A and B). The significant differences regarding to the control were marked by asterisks (*P<0.05; **P<0.01). ( C ) After transfection, the cells were stimulated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. PSF re-localization, p-Erk1/2, p-IKK α/β and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) and PSF in nuclear fractions were examined after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; PSF KD, HBMECs transfected with PSF siRNA; UNT: Untreated HBMECs. ( D ) Time course analysis of IbeA-induced tyrosine phosphorylation of PSF. HBMECs were incubated with the IbeA protein (0.1 µg/ml) for 2, 6, and 24 hrs, respectively. The cytoplasmic fractions were extracted and immunoprecipitated (IP) using the anti-phosphotyrosine antibody as described in . The Tyr-IP complexes were subjected to Western blot using the mouse monoclonal antibody against PSF. Total mouse IgG was detected as an internal loading control. CON: the IP control without primary antibody incubation; 0 h: the control HBMECs without IbeA incubation.

    Journal: PLoS ONE

    Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

    doi: 10.1371/journal.pone.0035862

    Figure Lengend Snippet: HBMECs were transfected with PSF or control siRNA as described in . IbeA+ E. coli K1 penetration ( A ) and PMN transmigration ( B ) across siRNA-transfected HBMECs were performed as described in the . Both invasion and PMN transmigration assays were performed in triplicates. Results for invasion are expressed as a relative percentage compared to the penetration rate of E44 in the siRNA control (CON) (100%). Results for PMN transmigration are expressed as the percentage of PMN transmigration of total PMNs. The control siRNA-transfected HBMECs infected with E44 and ZD1 were used as the controls (panels A and B). The significant differences regarding to the control were marked by asterisks (*P<0.05; **P<0.01). ( C ) After transfection, the cells were stimulated with E44 or ZD1 (10 7 /ml) for 30 min or 2 h. PSF re-localization, p-Erk1/2, p-IKK α/β and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) and PSF in nuclear fractions were examined after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; PSF KD, HBMECs transfected with PSF siRNA; UNT: Untreated HBMECs. ( D ) Time course analysis of IbeA-induced tyrosine phosphorylation of PSF. HBMECs were incubated with the IbeA protein (0.1 µg/ml) for 2, 6, and 24 hrs, respectively. The cytoplasmic fractions were extracted and immunoprecipitated (IP) using the anti-phosphotyrosine antibody as described in . The Tyr-IP complexes were subjected to Western blot using the mouse monoclonal antibody against PSF. Total mouse IgG was detected as an internal loading control. CON: the IP control without primary antibody incubation; 0 h: the control HBMECs without IbeA incubation.

    Article Snippet: Expression and purification of recombinant proteins in E. coli BL21 (DE3) cells were carried out according to the manufacturer's instructions (EMD Bioscience).

    Techniques: Transfection, Transmigration Assay, Infection, Incubation, Immunoprecipitation, Western Blot

    Immunoblotting analysis of polyubiquitinylated proteins (Ub-prs): ( A ) HBMECs with siRNA-mediated knockdown of vimentin; and ( B ) HBMECs transduced with the lentivirus constructs expressing GFP–VRT and GFP. In both (A) and (B), all the cells were incubated with or without E. coli K1 strains (E44 and ZD1, 10 7 /ml) for 2 h. The Ub-prs were detected in cytoplasmic fractions to determine the proteasomal degradation as described in . β-actin was used as an internal loading control. In all experiments, untreated HBMECs (UNT) were taken as controls.

    Journal: PLoS ONE

    Article Title: Vimentin and PSF Act in Concert to Regulate IbeA+ E. coli K1 Induced Activation and Nuclear Translocation of NF-κB in Human Brain Endothelial Cells

    doi: 10.1371/journal.pone.0035862

    Figure Lengend Snippet: Immunoblotting analysis of polyubiquitinylated proteins (Ub-prs): ( A ) HBMECs with siRNA-mediated knockdown of vimentin; and ( B ) HBMECs transduced with the lentivirus constructs expressing GFP–VRT and GFP. In both (A) and (B), all the cells were incubated with or without E. coli K1 strains (E44 and ZD1, 10 7 /ml) for 2 h. The Ub-prs were detected in cytoplasmic fractions to determine the proteasomal degradation as described in . β-actin was used as an internal loading control. In all experiments, untreated HBMECs (UNT) were taken as controls.

    Article Snippet: Expression and purification of recombinant proteins in E. coli BL21 (DE3) cells were carried out according to the manufacturer's instructions (EMD Bioscience).

    Techniques: Western Blot, Transduction, Construct, Expressing, Incubation

    Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of BL21 (DE3) pLysS (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS (pRSETC) as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).

    Journal: Biotechnology Research International

    Article Title: Display of the Viral Epitopes on Lactococcus lactis : A Model for Food Grade Vaccine against EV71

    doi: 10.1155/2013/431315

    Figure Lengend Snippet: Western blot analyses of the over-expressed recombinant fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ). Lane 1, Total protein of BL21 (DE3) pLysS (pSVacVP1 1-201nt ); lane 2, Total protein of BL21 (DE3) pLysS (pRSETC) as negative control; lane 3, Total protein of BL21 (DE3) pLysS (pSVacVP1 103-300nt ). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa), AcmA/VP1 35-100aa (~25 kDa).

    Article Snippet: Purified fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ) and total protein extractions of L. lactis and E. coli BL21 (DE3) pLysS (pRSETC) cells were separated by 12.5% SDS-PAGE and electroblotted on a PVDF (Millipore Corp., Billerica, MA, USA) membrane.

    Techniques: Western Blot, Recombinant, Negative Control

    Detection of serum antibody response against VP1 1-67aa and VP1 35-100aa epitopes of EV71 in mice immunized with L. lactis displaying AcmA/VP1 35-100aa . Lane 1: BSA protein; lane 2: E. coli (pRSET) total proteins; lane 3: total proteins of L. lactis MG1363; lane 4: purified AcmA/VP1 1-67aa proteins; lane 5: purified AcmA/VP1 35-100aa protein; lane M: protein marker (Fermentas, Hanover, MD, USA). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa) and AcmA/VP1 35-100aa (~25 kDa).

    Journal: Biotechnology Research International

    Article Title: Display of the Viral Epitopes on Lactococcus lactis : A Model for Food Grade Vaccine against EV71

    doi: 10.1155/2013/431315

    Figure Lengend Snippet: Detection of serum antibody response against VP1 1-67aa and VP1 35-100aa epitopes of EV71 in mice immunized with L. lactis displaying AcmA/VP1 35-100aa . Lane 1: BSA protein; lane 2: E. coli (pRSET) total proteins; lane 3: total proteins of L. lactis MG1363; lane 4: purified AcmA/VP1 1-67aa proteins; lane 5: purified AcmA/VP1 35-100aa protein; lane M: protein marker (Fermentas, Hanover, MD, USA). The arrow shows recombinant fusion proteins: AcmA/VP1 1-67aa (~28 kDa) and AcmA/VP1 35-100aa (~25 kDa).

    Article Snippet: Purified fusion proteins (AcmA/VP1 1-67aa and AcmA/VP1 35-100aa ) and total protein extractions of L. lactis and E. coli BL21 (DE3) pLysS (pRSETC) cells were separated by 12.5% SDS-PAGE and electroblotted on a PVDF (Millipore Corp., Billerica, MA, USA) membrane.

    Techniques: Purification, Marker, Recombinant