Structured Review

Polysciences inc pei
In vivo uptake of polyplexes by DCs in the draining lymph nodes 24 h after s.c. injection in mice. Polyplexes were prepared from <t>PEG-</t> b -PAEM 75 or <t>PEI</t> with anionic fluorescently-labeled dextran (Dex). For comparison, Dex alone and 100-nm fluorescent polystyrene
Pei, supplied by Polysciences inc, 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/pei/product/Polysciences inc
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99
pei - by Bioz Stars, 2021-07
86/100 stars

Images

1) Product Images from "Well-defined Block Copolymers for Gene Delivery to Dendritic Cells: Probing the Effect of Polycation Chain-length"

Article Title: Well-defined Block Copolymers for Gene Delivery to Dendritic Cells: Probing the Effect of Polycation Chain-length

Journal: Journal of controlled release : official journal of the Controlled Release Society

doi: 10.1016/j.jconrel.2009.10.021

In vivo uptake of polyplexes by DCs in the draining lymph nodes 24 h after s.c. injection in mice. Polyplexes were prepared from PEG- b -PAEM 75 or PEI with anionic fluorescently-labeled dextran (Dex). For comparison, Dex alone and 100-nm fluorescent polystyrene
Figure Legend Snippet: In vivo uptake of polyplexes by DCs in the draining lymph nodes 24 h after s.c. injection in mice. Polyplexes were prepared from PEG- b -PAEM 75 or PEI with anionic fluorescently-labeled dextran (Dex). For comparison, Dex alone and 100-nm fluorescent polystyrene

Techniques Used: In Vivo, Injection, Mouse Assay, Labeling

2) Product Images from "Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines"

Article Title: Comparative analysis of enzymatically produced novel linear DNA constructs with plasmids for use as DNA vaccines

Journal: Gene therapy

doi: 10.1038/gt.2014.37

Doggybone and plasmid have similar expression levels in vitro The efficiency of transfection of 1 μg Doggybone (●) or plasmid (□) encoding luciferase in CHO-K1 using electroporation (A), Lipofectamine (B), PEI (C) or Polyfect (D) was tested. Light emission was recorded as relative light units (RLU).The size (E) and the charge (F) of complexes formed by each transfection reagent at pH 7 was characterised by dynamic light scattering. Bars/points represent mean of n=3 replicates +/− SEM * p
Figure Legend Snippet: Doggybone and plasmid have similar expression levels in vitro The efficiency of transfection of 1 μg Doggybone (●) or plasmid (□) encoding luciferase in CHO-K1 using electroporation (A), Lipofectamine (B), PEI (C) or Polyfect (D) was tested. Light emission was recorded as relative light units (RLU).The size (E) and the charge (F) of complexes formed by each transfection reagent at pH 7 was characterised by dynamic light scattering. Bars/points represent mean of n=3 replicates +/− SEM * p

Techniques Used: Plasmid Preparation, Expressing, In Vitro, Transfection, Luciferase, Electroporation

3) Product Images from "Abstracts from the 25th European Society for Animal Cell Technology Meeting: Cell Technologies for Innovative Therapies"

Article Title: Abstracts from the 25th European Society for Animal Cell Technology Meeting: Cell Technologies for Innovative Therapies

Journal: BMC Proceedings

doi: 10.1186/s12919-018-0097-x

Production of Fab fragment under optimal conditions in a shake-flask culture. Cells at a density of 1 x 10 6 cells/cm 3 were transfected with 5 μg/(10 6 cells) of DNA (Hc:Lc gene ratio = 3:7) using 10 μg/(10 6 cells) of PEI in the serum-free medium PSFM-J1.Transfected cells were incubated at 24 °C. a Density of viable cells. Density of untransfected cells (open circles) is also shown. b Concentration of Fab fragments in the culture supernatant. Bars represent the means ± S.D. obtained from eight ( a ) or three ( b ) different determinations
Figure Legend Snippet: Production of Fab fragment under optimal conditions in a shake-flask culture. Cells at a density of 1 x 10 6 cells/cm 3 were transfected with 5 μg/(10 6 cells) of DNA (Hc:Lc gene ratio = 3:7) using 10 μg/(10 6 cells) of PEI in the serum-free medium PSFM-J1.Transfected cells were incubated at 24 °C. a Density of viable cells. Density of untransfected cells (open circles) is also shown. b Concentration of Fab fragments in the culture supernatant. Bars represent the means ± S.D. obtained from eight ( a ) or three ( b ) different determinations

Techniques Used: Transfection, Incubation, Concentration Assay

PEIpro® requires less reagent and similar to lower DNA amount compared to other PEIs. Suspension HEK-293 and CHO cells were seeded at 1×10 6 cells/mL in serum free medium and transfected with PEIpro®, PEI “Max” and L-PEI 25 kDa (Polysciences, Warrington, PA) resuspended at 1 mg/ml. Luciferase expression was assayed 48 h after transfection using a conventional luciferase assay
Figure Legend Snippet: PEIpro® requires less reagent and similar to lower DNA amount compared to other PEIs. Suspension HEK-293 and CHO cells were seeded at 1×10 6 cells/mL in serum free medium and transfected with PEIpro®, PEI “Max” and L-PEI 25 kDa (Polysciences, Warrington, PA) resuspended at 1 mg/ml. Luciferase expression was assayed 48 h after transfection using a conventional luciferase assay

Techniques Used: Transfection, Luciferase, Expressing

4) Product Images from "HIV-1 Vpr stimulates NF-?B and AP-1 signaling by activating TAK1"

Article Title: HIV-1 Vpr stimulates NF-?B and AP-1 signaling by activating TAK1

Journal: Retrovirology

doi: 10.1186/1742-4690-11-45

The S79A mutant of Vpr is unable to activate NF-κB and AP-1. (A) Vpr S79A failed to associate with endogenous TAK1. HEK293T cells (4 × 10 6 ) were transfected with wild type Flag-Vpr or its mutant S79A. After forty-eight hours, cell lysates were immunoprecipitated with anti-Flag antibody. Samples from both cell lysates and immunoprecipitates were probed with rabbit anti-TAK1 and anti-Flag antibodies. (B) Vpr S79A was unable to enhance the phosphorylation of TAK1. A total of 0.5 × 10 6 HEK293T cells were transfected with wild type Flag-Vpr or its mutant S79A. After forty-eight hours, cells were pretreated with 20 nM Calyculin A for 5 min. Whole cell lysates were subjected to Western blotting and probed with indicated antibodies. (C) HEK293T cells (4 × 10 6 ) were transfected with 1 μg pVSV-G along with 8 μg NLENY1-ES (WT), NLENY1-ΔVpr (ΔVpr), or NLENY1-S79A (S79A) by PEI. The cell lysates and viral supernatants were subjected to Western blotting with the indicated antibodies. (D) Jurkat cells (2 × 10 6 ) were infected with VSV-G pseudotyped WT, ΔVpr, or S79A equivalent to 500 ng p24 in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After two hours, cells were pretreated with 15 nM Calyculin A for 5 min. Whole cell lysates were subjected to Western blotting with the indicated antibodies. (E) Ser-79 is required for Vpr-induced NF-κB, AP-1, and HIV-1 LTR activation. HeLa cells (0.1 × 10 6 ) were transfected with Flag-Vpr or its S79A mutant along with κB, AP-1, or HIV-1 LTR luciferase reporter plasmid. After forty-eight hours, luciferase activities were measured. The results shown are the averages of three independent experiments. The error bars indicate standard deviations. * P
Figure Legend Snippet: The S79A mutant of Vpr is unable to activate NF-κB and AP-1. (A) Vpr S79A failed to associate with endogenous TAK1. HEK293T cells (4 × 10 6 ) were transfected with wild type Flag-Vpr or its mutant S79A. After forty-eight hours, cell lysates were immunoprecipitated with anti-Flag antibody. Samples from both cell lysates and immunoprecipitates were probed with rabbit anti-TAK1 and anti-Flag antibodies. (B) Vpr S79A was unable to enhance the phosphorylation of TAK1. A total of 0.5 × 10 6 HEK293T cells were transfected with wild type Flag-Vpr or its mutant S79A. After forty-eight hours, cells were pretreated with 20 nM Calyculin A for 5 min. Whole cell lysates were subjected to Western blotting and probed with indicated antibodies. (C) HEK293T cells (4 × 10 6 ) were transfected with 1 μg pVSV-G along with 8 μg NLENY1-ES (WT), NLENY1-ΔVpr (ΔVpr), or NLENY1-S79A (S79A) by PEI. The cell lysates and viral supernatants were subjected to Western blotting with the indicated antibodies. (D) Jurkat cells (2 × 10 6 ) were infected with VSV-G pseudotyped WT, ΔVpr, or S79A equivalent to 500 ng p24 in the presence of 5 μg/ml polybrene by spinoculation at 300 xg for 30 min. After two hours, cells were pretreated with 15 nM Calyculin A for 5 min. Whole cell lysates were subjected to Western blotting with the indicated antibodies. (E) Ser-79 is required for Vpr-induced NF-κB, AP-1, and HIV-1 LTR activation. HeLa cells (0.1 × 10 6 ) were transfected with Flag-Vpr or its S79A mutant along with κB, AP-1, or HIV-1 LTR luciferase reporter plasmid. After forty-eight hours, luciferase activities were measured. The results shown are the averages of three independent experiments. The error bars indicate standard deviations. * P

Techniques Used: Mutagenesis, Transfection, Immunoprecipitation, Western Blot, Infection, Activation Assay, Luciferase, Plasmid Preparation

5) Product Images from "Constitutive activation of breast tumor kinase accelerates cell migration and tumor growth in vivo"

Article Title: Constitutive activation of breast tumor kinase accelerates cell migration and tumor growth in vivo

Journal: Oncogenesis

doi: 10.1038/oncsis.2012.11

Stable knockdown of BRK significantly suppresses migration of breast cancer cells in wound-healing assays. Stable BRK knockdown were performed on parental breast cancer cell lines, BT20 and SKBR3 using shRNA lentiviral vector plasmids from Santa Cruz biotechnology according to the manufacture's protocol. ( a , d ) Efficient knockdown of BRK in breast cancer cells. BT20 and SKBR3 cells were transfected with a GFP-expressing plasmid or control shRNA plasmid or an shRNA-BRK plasmid using the PEI transfection method (See ‘Materials and methods'). The GFP-control plasmid (lane 1, right and left panels) allowed the confirmation of the transduction efficiency by expressing GFP, detectable by fluorescence microscopy. The control shRNA plasmid (lanes 2) encode a scrambled shRNA sequence, which does not lead to mRNA degradation. BRK-shRNA lentiviral vector plasmid (lanes 3) comprises at least three lentiviral vector plasmids target-specific 19–25 nucleotides in shRNAs designed to knockdown BRK gene expression. Transfected cells were selected using puromycin. ( b , e ) BRK knockdown significantly suppresses migration of both BT20 and SKBR cells. The stable knockdown cells were analyzed for cell migration using the wound-healing assay in 6-well plates as described in Figure 3 legend. ( c , f ) The open area (scratch) was quantified with TScratch software. 49 The P -values were set at *** P ⩽0.0001 for statistically significance.
Figure Legend Snippet: Stable knockdown of BRK significantly suppresses migration of breast cancer cells in wound-healing assays. Stable BRK knockdown were performed on parental breast cancer cell lines, BT20 and SKBR3 using shRNA lentiviral vector plasmids from Santa Cruz biotechnology according to the manufacture's protocol. ( a , d ) Efficient knockdown of BRK in breast cancer cells. BT20 and SKBR3 cells were transfected with a GFP-expressing plasmid or control shRNA plasmid or an shRNA-BRK plasmid using the PEI transfection method (See ‘Materials and methods'). The GFP-control plasmid (lane 1, right and left panels) allowed the confirmation of the transduction efficiency by expressing GFP, detectable by fluorescence microscopy. The control shRNA plasmid (lanes 2) encode a scrambled shRNA sequence, which does not lead to mRNA degradation. BRK-shRNA lentiviral vector plasmid (lanes 3) comprises at least three lentiviral vector plasmids target-specific 19–25 nucleotides in shRNAs designed to knockdown BRK gene expression. Transfected cells were selected using puromycin. ( b , e ) BRK knockdown significantly suppresses migration of both BT20 and SKBR cells. The stable knockdown cells were analyzed for cell migration using the wound-healing assay in 6-well plates as described in Figure 3 legend. ( c , f ) The open area (scratch) was quantified with TScratch software. 49 The P -values were set at *** P ⩽0.0001 for statistically significance.

Techniques Used: Migration, shRNA, Plasmid Preparation, Transfection, Expressing, Transduction, Fluorescence, Microscopy, Sequencing, Wound Healing Assay, Software

Tyr447Phe BRK mutant is significantly more active than the WT BRK. ( a ) Schematic representation of BRK. The diagram shows the functional domains and the positions of some of the keep residues mutated in this study.( b ) Activity of BRK and BRK mutants in transfected HEK293 cells. WT BRK and BRK mutants, non-tagged and GFP-tagged, were transfected and expressed in HEK293 cells as described in ‘Materials and methods'. The cells were transiently transfected with 0.1% PEI ‘Max' (Polysciences Inc.) at a ratio of 3:1 reagent to DNA with the total amount of DNA being 2.5 μg per well in six-well dishes. Cells were washed 4 h after transfection, then cultured in DMEM containing 10% fetal calf serum for an additional 24 h. Cell lysates were subjected to SDS–PAGE and the proteins transferred onto nitrocellulose membranes and immunoblotted with antiphosphotyrosine antibody (PY20) and anti-BRK and anti-phospho-BRK (pTyr342). Anti-β-tubulin served as a loading control.
Figure Legend Snippet: Tyr447Phe BRK mutant is significantly more active than the WT BRK. ( a ) Schematic representation of BRK. The diagram shows the functional domains and the positions of some of the keep residues mutated in this study.( b ) Activity of BRK and BRK mutants in transfected HEK293 cells. WT BRK and BRK mutants, non-tagged and GFP-tagged, were transfected and expressed in HEK293 cells as described in ‘Materials and methods'. The cells were transiently transfected with 0.1% PEI ‘Max' (Polysciences Inc.) at a ratio of 3:1 reagent to DNA with the total amount of DNA being 2.5 μg per well in six-well dishes. Cells were washed 4 h after transfection, then cultured in DMEM containing 10% fetal calf serum for an additional 24 h. Cell lysates were subjected to SDS–PAGE and the proteins transferred onto nitrocellulose membranes and immunoblotted with antiphosphotyrosine antibody (PY20) and anti-BRK and anti-phospho-BRK (pTyr342). Anti-β-tubulin served as a loading control.

Techniques Used: Mutagenesis, Functional Assay, Activity Assay, Transfection, Cell Culture, SDS Page

6) Product Images from "Generation and Characterization of the First Immortalized Alpaca Cell Line Suitable for Diagnostic and Immunization Studies"

Article Title: Generation and Characterization of the First Immortalized Alpaca Cell Line Suitable for Diagnostic and Immunization Studies

Journal: PLoS ONE

doi: 10.1371/journal.pone.0105643

Transfection. Representative phase contrast and fluorescence images (10x) of ASSCs transfected with a GFP expression plasmid by different methods (Electroporation, LTX, PEI and Fugene HD), along with the efficiency of transfection as measured by cytometry. Each set of transfections was performed three times and standard deviations were negligible.
Figure Legend Snippet: Transfection. Representative phase contrast and fluorescence images (10x) of ASSCs transfected with a GFP expression plasmid by different methods (Electroporation, LTX, PEI and Fugene HD), along with the efficiency of transfection as measured by cytometry. Each set of transfections was performed three times and standard deviations were negligible.

Techniques Used: Transfection, Fluorescence, Expressing, Plasmid Preparation, Electroporation, Cytometry

7) Product Images from "Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest"

Article Title: Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest

Journal: The EMBO Journal

doi: 10.15252/embj.201797877

Chk1 inhibits the transcriptional repressor function of E2F7 and E2F8 to promote cell cycle progression and prevent apoptosis Left panel shows a schematic view of the experimental setting. HeLa/TO cells were synchronized with hydroxyurea (HU) for 16 h, then released by washing and adding new medium containing doxycycline (Dox) to induce expression of the transgenes. Cell lines incubated with medium without doxycycline (Vehicle, Veh) were used as controls. Expression of WT and Chk1 mutant versions of E2F7/8 was monitored by EGFP fluorescence, DNA was visualized by adding fluorescent SiR‐DNA to the medium, and cell morphology was evaluated by differential interference contrast (DIC). Per condition, 100 individual cells were traced. Each cell was followed until it successfully finished mitosis and divided into two daughter cells, for a maximum of 24 h. The graphs on the right show the quantification of the mitotic events during live cell imaging. Scale bar: 10 μm. Quantification of apoptosis by flow cytometric analysis of Annexin‐V staining. Experiments were performed as outlined in (A), and cells were harvested for flow cytometry 24 h after HU release. Apoptotic cells were counted as Annexin‐positive and DAPI‐negative. Quantitative PCR of CDC6 , RAD51 , CDC25A, and CCNE1 expression in HeLa/TO cell lines expressing wild‐type and mutant versions of E2F7/8. Chk1 phosphorylation does not change the promoter enrichment of E2F7 and E2F8. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids tagged with GFP. 48 h after transfection, cells were harvested for chromatin immunoprecipitation (ChIP) followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. Data information: In (B–D), data represent average ± SEM ( n = 3); * P
Figure Legend Snippet: Chk1 inhibits the transcriptional repressor function of E2F7 and E2F8 to promote cell cycle progression and prevent apoptosis Left panel shows a schematic view of the experimental setting. HeLa/TO cells were synchronized with hydroxyurea (HU) for 16 h, then released by washing and adding new medium containing doxycycline (Dox) to induce expression of the transgenes. Cell lines incubated with medium without doxycycline (Vehicle, Veh) were used as controls. Expression of WT and Chk1 mutant versions of E2F7/8 was monitored by EGFP fluorescence, DNA was visualized by adding fluorescent SiR‐DNA to the medium, and cell morphology was evaluated by differential interference contrast (DIC). Per condition, 100 individual cells were traced. Each cell was followed until it successfully finished mitosis and divided into two daughter cells, for a maximum of 24 h. The graphs on the right show the quantification of the mitotic events during live cell imaging. Scale bar: 10 μm. Quantification of apoptosis by flow cytometric analysis of Annexin‐V staining. Experiments were performed as outlined in (A), and cells were harvested for flow cytometry 24 h after HU release. Apoptotic cells were counted as Annexin‐positive and DAPI‐negative. Quantitative PCR of CDC6 , RAD51 , CDC25A, and CCNE1 expression in HeLa/TO cell lines expressing wild‐type and mutant versions of E2F7/8. Chk1 phosphorylation does not change the promoter enrichment of E2F7 and E2F8. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids tagged with GFP. 48 h after transfection, cells were harvested for chromatin immunoprecipitation (ChIP) followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. Data information: In (B–D), data represent average ± SEM ( n = 3); * P

Techniques Used: Expressing, Incubation, Mutagenesis, Fluorescence, Live Cell Imaging, Flow Cytometry, Staining, Cytometry, Real-time Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation

14‐3‐3 ζ mediates Chk1‐dependent inhibition of E2F7/8 Scatter plot shows the relative enrichment scores of common E2F7 and E2F8 binding partners. Red dashed line indicates the fold change cutoff ( > 2.0). 14‐3‐3 isoforms are highlighted as red dots. 14‐3‐3 ζ interacts with wild‐type E2F7/8 but not with alanine mutants in vitro . HEK 293T cells were transfected with indicated constructs, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against Myc or GFP. Inputs represent loading controls. Chromatin immunoprecipitation (ChIP) demonstrated that 14‐3‐3 proteins bind to the E2F7/8 target gene promoters. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids. 48 h after transfection, cells were harvested for ChIP assay and followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. A primer set designed against a distal region in the E2F1 gene served as a negative control. Luciferase reporter assay in U2OS cells using E2F1 promoter plasmid. Plasmids were co‐transfected either with wild‐type or alanine mutant, either alone or with 14‐3‐3 ζ plasmid. 14‐3‐3 inhibitor BV‐02 disrupts the interaction between 14‐3‐3 ζ and E2F7/8. Transfection was carried out as shown, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against GFP or Myc. Inputs represent loading controls. Transcript levels of common E2F7 and E2F8 target genes after treatment with HU or HU + BV‐02 for 16 h, determined by qPCR. Data information: In (C, D and F), data represent average ± SEM ( n = 3); * P
Figure Legend Snippet: 14‐3‐3 ζ mediates Chk1‐dependent inhibition of E2F7/8 Scatter plot shows the relative enrichment scores of common E2F7 and E2F8 binding partners. Red dashed line indicates the fold change cutoff ( > 2.0). 14‐3‐3 isoforms are highlighted as red dots. 14‐3‐3 ζ interacts with wild‐type E2F7/8 but not with alanine mutants in vitro . HEK 293T cells were transfected with indicated constructs, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against Myc or GFP. Inputs represent loading controls. Chromatin immunoprecipitation (ChIP) demonstrated that 14‐3‐3 proteins bind to the E2F7/8 target gene promoters. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids. 48 h after transfection, cells were harvested for ChIP assay and followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. A primer set designed against a distal region in the E2F1 gene served as a negative control. Luciferase reporter assay in U2OS cells using E2F1 promoter plasmid. Plasmids were co‐transfected either with wild‐type or alanine mutant, either alone or with 14‐3‐3 ζ plasmid. 14‐3‐3 inhibitor BV‐02 disrupts the interaction between 14‐3‐3 ζ and E2F7/8. Transfection was carried out as shown, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against GFP or Myc. Inputs represent loading controls. Transcript levels of common E2F7 and E2F8 target genes after treatment with HU or HU + BV‐02 for 16 h, determined by qPCR. Data information: In (C, D and F), data represent average ± SEM ( n = 3); * P

Techniques Used: Inhibition, Binding Assay, In Vitro, Transfection, Construct, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Luciferase, Reporter Assay, Plasmid Preparation, Mutagenesis

14‐3‐3 proteins interact with E2F7/8 at the promoter regions Experimental procedures of the SILAC assay to identify binding partners of E2F7 and E2F8. 14‐3‐3 ε interacts with wild‐type but not alanine mutant E2F7 and E2F8 in vitro . HEK 293T cells were transfected as indicated, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against GFP or Myc. Inputs confirm similar expression of Myc‐14‐3‐3 ε in all samples. 14‐3‐3 ζ interacts with endogenous phosphorylated E2F7 and E2F8 in vivo . HEK 293T cells were transfected with Myc‐14‐3‐3 ζ and treated with or without HU. Lysates were precipitated with anti‐Myc antibodies and immunoblotted with antibodies directed against the Chk1 phosphorylation site of E2F7 S410 or E2F8 S396 (asterisks indicate the signal). Inputs confirm similar expression of Myc‐14‐3‐3 ζ in all samples. 14‐3‐3 ζ does not alter the nuclear localization of E2F7/8. U2OS cells were transfected with Myc‐14‐3‐3 ζ with either empty vector, EGFP wild‐type E2F7 or E2F8 followed by immunofluorescence staining. DAPI was used to stain nuclear DNA. Scale bar: 5 μm. Chromatin immunoprecipitation (ChIP) demonstrated that 14‐3‐3 proteins bind to the E2F7/8 target gene promoters. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids. 48 h after transfection, cells were harvested for ChIP assay and followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. A primer set designed against a distal region in the E2F1 gene served as a negative control. Validation of E2F7 and E2F8 knockdown efficiency of experiments shown in Fig 5 F. E2F7 and E2F8 mRNA levels was determined by qPCR. Data information: In (D, F), data represent mean ± SEM ( n = 3); ** P
Figure Legend Snippet: 14‐3‐3 proteins interact with E2F7/8 at the promoter regions Experimental procedures of the SILAC assay to identify binding partners of E2F7 and E2F8. 14‐3‐3 ε interacts with wild‐type but not alanine mutant E2F7 and E2F8 in vitro . HEK 293T cells were transfected as indicated, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against GFP or Myc. Inputs confirm similar expression of Myc‐14‐3‐3 ε in all samples. 14‐3‐3 ζ interacts with endogenous phosphorylated E2F7 and E2F8 in vivo . HEK 293T cells were transfected with Myc‐14‐3‐3 ζ and treated with or without HU. Lysates were precipitated with anti‐Myc antibodies and immunoblotted with antibodies directed against the Chk1 phosphorylation site of E2F7 S410 or E2F8 S396 (asterisks indicate the signal). Inputs confirm similar expression of Myc‐14‐3‐3 ζ in all samples. 14‐3‐3 ζ does not alter the nuclear localization of E2F7/8. U2OS cells were transfected with Myc‐14‐3‐3 ζ with either empty vector, EGFP wild‐type E2F7 or E2F8 followed by immunofluorescence staining. DAPI was used to stain nuclear DNA. Scale bar: 5 μm. Chromatin immunoprecipitation (ChIP) demonstrated that 14‐3‐3 proteins bind to the E2F7/8 target gene promoters. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids. 48 h after transfection, cells were harvested for ChIP assay and followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. A primer set designed against a distal region in the E2F1 gene served as a negative control. Validation of E2F7 and E2F8 knockdown efficiency of experiments shown in Fig 5 F. E2F7 and E2F8 mRNA levels was determined by qPCR. Data information: In (D, F), data represent mean ± SEM ( n = 3); ** P

Techniques Used: Binding Assay, Mutagenesis, In Vitro, Transfection, Expressing, In Vivo, Plasmid Preparation, Immunofluorescence, Staining, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control

8) Product Images from "Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction"

Article Title: Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction

Journal: World Journal of Stem Cells

doi: 10.4252/wjsc.v7.i1.223

Chemical transfection of fetal mesenchymal stem cells. Representative merged photomicrographs for (A-D) FuGENE 6, (E-H) Lipofectamine and (I-J) PEI mediated transfection of pcDNA3-eGFP into fMSCs at 3:1, 3:2, 6:1, and 5:2 reagent (μL): DNA (μg)
Figure Legend Snippet: Chemical transfection of fetal mesenchymal stem cells. Representative merged photomicrographs for (A-D) FuGENE 6, (E-H) Lipofectamine and (I-J) PEI mediated transfection of pcDNA3-eGFP into fMSCs at 3:1, 3:2, 6:1, and 5:2 reagent (μL): DNA (μg)

Techniques Used: Transfection

9) Product Images from "The transient expression of CHIKV VLP in large stirred tank bioreactors"

Article Title: The transient expression of CHIKV VLP in large stirred tank bioreactors

Journal: Cytotechnology

doi: 10.1007/s10616-019-00346-x

The effect of pH, inoculum source, and DNA–PEI concentration on CHIKV VLP production in the first ambr® 15 bioreactor study. Product titer was normalized to that of Vessel 1 condition (DNA 20 mg/L–PEI 40 mg/L at pH 7.3 ± 0.2) on Day 4. Vessel 16 had a control issue during the study and its data was not included. E: early exponential; M: middle exponential; L: late exponential; S: stationary
Figure Legend Snippet: The effect of pH, inoculum source, and DNA–PEI concentration on CHIKV VLP production in the first ambr® 15 bioreactor study. Product titer was normalized to that of Vessel 1 condition (DNA 20 mg/L–PEI 40 mg/L at pH 7.3 ± 0.2) on Day 4. Vessel 16 had a control issue during the study and its data was not included. E: early exponential; M: middle exponential; L: late exponential; S: stationary

Techniques Used: Concentration Assay

The effect of pH and DNA–PEI concentration on cell growth in the second ambr® 15 bioreactor study. The first cell counts were taken after the addition of 1.5× CDM4HEK293 production medium
Figure Legend Snippet: The effect of pH and DNA–PEI concentration on cell growth in the second ambr® 15 bioreactor study. The first cell counts were taken after the addition of 1.5× CDM4HEK293 production medium

Techniques Used: Concentration Assay

The toxicity of DNA, PEI, and combination of DNA and PEI measured by cellular viability in the third ambr® 15 bioreactor study. Samples were taken 3 h after transfection. Inoculum cells had a viability of more than 95%
Figure Legend Snippet: The toxicity of DNA, PEI, and combination of DNA and PEI measured by cellular viability in the third ambr® 15 bioreactor study. Samples were taken 3 h after transfection. Inoculum cells had a viability of more than 95%

Techniques Used: Transfection

The effect of pH and DNA–PEI concentration on CHIKV VLP production in the second ambr® 15 bioreactor study. Product titer was normalized to that of the control condition DNA 20 mg/L–PEI 40 mg/L on Day 4
Figure Legend Snippet: The effect of pH and DNA–PEI concentration on CHIKV VLP production in the second ambr® 15 bioreactor study. Product titer was normalized to that of the control condition DNA 20 mg/L–PEI 40 mg/L on Day 4

Techniques Used: Concentration Assay

10) Product Images from "Constitutive activation of breast tumor kinase accelerates cell migration and tumor growth in vivo"

Article Title: Constitutive activation of breast tumor kinase accelerates cell migration and tumor growth in vivo

Journal: Oncogenesis

doi: 10.1038/oncsis.2012.11

Stable knockdown of BRK significantly suppresses migration of breast cancer cells in wound-healing assays. Stable BRK knockdown were performed on parental breast cancer cell lines, BT20 and SKBR3 using shRNA lentiviral vector plasmids from Santa Cruz biotechnology according to the manufacture's protocol. ( a , d ) Efficient knockdown of BRK in breast cancer cells. BT20 and SKBR3 cells were transfected with a GFP-expressing plasmid or control shRNA plasmid or an shRNA-BRK plasmid using the PEI transfection method (See ‘Materials and methods'). The GFP-control plasmid (lane 1, right and left panels) allowed the confirmation of the transduction efficiency by expressing GFP, detectable by fluorescence microscopy. The control shRNA plasmid (lanes 2) encode a scrambled shRNA sequence, which does not lead to mRNA degradation. BRK-shRNA lentiviral vector plasmid (lanes 3) comprises at least three lentiviral vector plasmids target-specific 19–25 nucleotides in shRNAs designed to knockdown BRK gene expression. Transfected cells were selected using puromycin. ( b , e ) BRK knockdown significantly suppresses migration of both BT20 and SKBR cells. The stable knockdown cells were analyzed for cell migration using the wound-healing assay in 6-well plates as described in Figure 3 legend. ( c , f ) The open area (scratch) was quantified with TScratch software. 49 The P -values were set at *** P ⩽0.0001 for statistically significance.
Figure Legend Snippet: Stable knockdown of BRK significantly suppresses migration of breast cancer cells in wound-healing assays. Stable BRK knockdown were performed on parental breast cancer cell lines, BT20 and SKBR3 using shRNA lentiviral vector plasmids from Santa Cruz biotechnology according to the manufacture's protocol. ( a , d ) Efficient knockdown of BRK in breast cancer cells. BT20 and SKBR3 cells were transfected with a GFP-expressing plasmid or control shRNA plasmid or an shRNA-BRK plasmid using the PEI transfection method (See ‘Materials and methods'). The GFP-control plasmid (lane 1, right and left panels) allowed the confirmation of the transduction efficiency by expressing GFP, detectable by fluorescence microscopy. The control shRNA plasmid (lanes 2) encode a scrambled shRNA sequence, which does not lead to mRNA degradation. BRK-shRNA lentiviral vector plasmid (lanes 3) comprises at least three lentiviral vector plasmids target-specific 19–25 nucleotides in shRNAs designed to knockdown BRK gene expression. Transfected cells were selected using puromycin. ( b , e ) BRK knockdown significantly suppresses migration of both BT20 and SKBR cells. The stable knockdown cells were analyzed for cell migration using the wound-healing assay in 6-well plates as described in Figure 3 legend. ( c , f ) The open area (scratch) was quantified with TScratch software. 49 The P -values were set at *** P ⩽0.0001 for statistically significance.

Techniques Used: Migration, shRNA, Plasmid Preparation, Transfection, Expressing, Transduction, Fluorescence, Microscopy, Sequencing, Wound Healing Assay, Software

Tyr447Phe BRK mutant is significantly more active than the WT BRK. ( a ) Schematic representation of BRK. The diagram shows the functional domains and the positions of some of the keep residues mutated in this study.( b ) Activity of BRK and BRK mutants in transfected HEK293 cells. WT BRK and BRK mutants, non-tagged and GFP-tagged, were transfected and expressed in HEK293 cells as described in ‘Materials and methods'. The cells were transiently transfected with 0.1% PEI ‘Max' (Polysciences Inc.) at a ratio of 3:1 reagent to DNA with the total amount of DNA being 2.5 μg per well in six-well dishes. Cells were washed 4 h after transfection, then cultured in DMEM containing 10% fetal calf serum for an additional 24 h. Cell lysates were subjected to SDS–PAGE and the proteins transferred onto nitrocellulose membranes and immunoblotted with antiphosphotyrosine antibody (PY20) and anti-BRK and anti-phospho-BRK (pTyr342). Anti-β-tubulin served as a loading control.
Figure Legend Snippet: Tyr447Phe BRK mutant is significantly more active than the WT BRK. ( a ) Schematic representation of BRK. The diagram shows the functional domains and the positions of some of the keep residues mutated in this study.( b ) Activity of BRK and BRK mutants in transfected HEK293 cells. WT BRK and BRK mutants, non-tagged and GFP-tagged, were transfected and expressed in HEK293 cells as described in ‘Materials and methods'. The cells were transiently transfected with 0.1% PEI ‘Max' (Polysciences Inc.) at a ratio of 3:1 reagent to DNA with the total amount of DNA being 2.5 μg per well in six-well dishes. Cells were washed 4 h after transfection, then cultured in DMEM containing 10% fetal calf serum for an additional 24 h. Cell lysates were subjected to SDS–PAGE and the proteins transferred onto nitrocellulose membranes and immunoblotted with antiphosphotyrosine antibody (PY20) and anti-BRK and anti-phospho-BRK (pTyr342). Anti-β-tubulin served as a loading control.

Techniques Used: Mutagenesis, Functional Assay, Activity Assay, Transfection, Cell Culture, SDS Page

11) Product Images from "Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest"

Article Title: Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest

Journal: The EMBO Journal

doi: 10.15252/embj.201797877

Chk1 inhibits the transcriptional repressor function of E2F7 and E2F8 to promote cell cycle progression and prevent apoptosis A Left panel shows a schematic view of the experimental setting. HeLa/TO cells were synchronized with hydroxyurea (HU) for 16 h, then released by washing and adding new medium containing doxycycline (Dox) to induce expression of the transgenes. Cell lines incubated with medium without doxycycline (Vehicle, Veh) were used as controls. Expression of WT and Chk1 mutant versions of E2F7/8 was monitored by EGFP fluorescence, DNA was visualized by adding fluorescent SiR‐DNA to the medium, and cell morphology was evaluated by differential interference contrast (DIC). Per condition, 100 individual cells were traced. Each cell was followed until it successfully finished mitosis and divided into two daughter cells, for a maximum of 24 h. The graphs on the right show the quantification of the mitotic events during live cell imaging. Scale bar: 10 μm. B Quantification of apoptosis by flow cytometric analysis of Annexin‐V staining. Experiments were performed as outlined in (A), and cells were harvested for flow cytometry 24 h after HU release. Apoptotic cells were counted as Annexin‐positive and DAPI‐negative. C, D Quantitative PCR of CDC6 , RAD51 , CDC25A, and CCNE1 expression in HeLa/TO cell lines expressing wild‐type and mutant versions of E2F7/8. E Chk1 phosphorylation does not change the promoter enrichment of E2F7 and E2F8. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids tagged with GFP. 48 h after transfection, cells were harvested for chromatin immunoprecipitation (ChIP) followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. Data information: In (B–D), data represent average ± SEM ( n = 3); * P
Figure Legend Snippet: Chk1 inhibits the transcriptional repressor function of E2F7 and E2F8 to promote cell cycle progression and prevent apoptosis A Left panel shows a schematic view of the experimental setting. HeLa/TO cells were synchronized with hydroxyurea (HU) for 16 h, then released by washing and adding new medium containing doxycycline (Dox) to induce expression of the transgenes. Cell lines incubated with medium without doxycycline (Vehicle, Veh) were used as controls. Expression of WT and Chk1 mutant versions of E2F7/8 was monitored by EGFP fluorescence, DNA was visualized by adding fluorescent SiR‐DNA to the medium, and cell morphology was evaluated by differential interference contrast (DIC). Per condition, 100 individual cells were traced. Each cell was followed until it successfully finished mitosis and divided into two daughter cells, for a maximum of 24 h. The graphs on the right show the quantification of the mitotic events during live cell imaging. Scale bar: 10 μm. B Quantification of apoptosis by flow cytometric analysis of Annexin‐V staining. Experiments were performed as outlined in (A), and cells were harvested for flow cytometry 24 h after HU release. Apoptotic cells were counted as Annexin‐positive and DAPI‐negative. C, D Quantitative PCR of CDC6 , RAD51 , CDC25A, and CCNE1 expression in HeLa/TO cell lines expressing wild‐type and mutant versions of E2F7/8. E Chk1 phosphorylation does not change the promoter enrichment of E2F7 and E2F8. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids tagged with GFP. 48 h after transfection, cells were harvested for chromatin immunoprecipitation (ChIP) followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. Data information: In (B–D), data represent average ± SEM ( n = 3); * P

Techniques Used: Expressing, Incubation, Mutagenesis, Fluorescence, Live Cell Imaging, Flow Cytometry, Staining, Cytometry, Real-time Polymerase Chain Reaction, Transfection, Chromatin Immunoprecipitation

14‐3‐3 ζ mediates Chk1‐dependent inhibition of E2F7/8 Scatter plot shows the relative enrichment scores of common E2F7 and E2F8 binding partners. Red dashed line indicates the fold change cutoff ( > 2.0). 14‐3‐3 isoforms are highlighted as red dots. 14‐3‐3 ζ interacts with wild‐type E2F7/8 but not with alanine mutants in vitro . HEK 293T cells were transfected with indicated constructs, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against Myc or GFP. Inputs represent loading controls. Chromatin immunoprecipitation (ChIP) demonstrated that 14‐3‐3 proteins bind to the E2F7/8 target gene promoters. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids. 48 h after transfection, cells were harvested for ChIP assay and followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. A primer set designed against a distal region in the E2F1 gene served as a negative control. Luciferase reporter assay in U2OS cells using E2F1 promoter plasmid. Plasmids were co‐transfected either with wild‐type or alanine mutant, either alone or with 14‐3‐3 ζ plasmid. 14‐3‐3 inhibitor BV‐02 disrupts the interaction between 14‐3‐3 ζ and E2F7/8. Transfection was carried out as shown, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against GFP or Myc. Inputs represent loading controls. Transcript levels of common E2F7 and E2F8 target genes after treatment with HU or HU + BV‐02 for 16 h, determined by qPCR. Data information: In (C, D and F), data represent average ± SEM ( n = 3); * P
Figure Legend Snippet: 14‐3‐3 ζ mediates Chk1‐dependent inhibition of E2F7/8 Scatter plot shows the relative enrichment scores of common E2F7 and E2F8 binding partners. Red dashed line indicates the fold change cutoff ( > 2.0). 14‐3‐3 isoforms are highlighted as red dots. 14‐3‐3 ζ interacts with wild‐type E2F7/8 but not with alanine mutants in vitro . HEK 293T cells were transfected with indicated constructs, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against Myc or GFP. Inputs represent loading controls. Chromatin immunoprecipitation (ChIP) demonstrated that 14‐3‐3 proteins bind to the E2F7/8 target gene promoters. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids. 48 h after transfection, cells were harvested for ChIP assay and followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters. A primer set designed against a distal region in the E2F1 gene served as a negative control. Luciferase reporter assay in U2OS cells using E2F1 promoter plasmid. Plasmids were co‐transfected either with wild‐type or alanine mutant, either alone or with 14‐3‐3 ζ plasmid. 14‐3‐3 inhibitor BV‐02 disrupts the interaction between 14‐3‐3 ζ and E2F7/8. Transfection was carried out as shown, and lysates were precipitated with GFP‐Trap beads and immunoblotted with antibodies directed against GFP or Myc. Inputs represent loading controls. Transcript levels of common E2F7 and E2F8 target genes after treatment with HU or HU + BV‐02 for 16 h, determined by qPCR. Data information: In (C, D and F), data represent average ± SEM ( n = 3); * P

Techniques Used: Inhibition, Binding Assay, In Vitro, Transfection, Construct, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Luciferase, Reporter Assay, Plasmid Preparation, Mutagenesis

Related Articles

Live Cell Imaging:

Article Title: Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation
Article Snippet: .. Live cell imaging Plasmid (pEYFP-Nuc [a gift of Dr. T. Inoue], 0.25 µg) and pmCherry-SAMHD1 -HA (2 µg) were transfected into HEK293T cells using PEI Max (Polysciences) according to the manufacturer's protocol. .. For live cell imaging, HEK293T cells were transfected in 6-well coverslip glass-bottomed cell culture dishes (InVitro Scientific) when the cells were ∼80% confluent, and then visualized after 24 h using a Zeiss LSM510-Meta confocal imaging system equipped with four argon lasers (458-, 477-, 488-, and 514-nm lines), two HeNe lasers (542 and 633 nm), and one diode laser (405 nm).

Plasmid Preparation:

Article Title: Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation
Article Snippet: .. Live cell imaging Plasmid (pEYFP-Nuc [a gift of Dr. T. Inoue], 0.25 µg) and pmCherry-SAMHD1 -HA (2 µg) were transfected into HEK293T cells using PEI Max (Polysciences) according to the manufacturer's protocol. .. For live cell imaging, HEK293T cells were transfected in 6-well coverslip glass-bottomed cell culture dishes (InVitro Scientific) when the cells were ∼80% confluent, and then visualized after 24 h using a Zeiss LSM510-Meta confocal imaging system equipped with four argon lasers (458-, 477-, 488-, and 514-nm lines), two HeNe lasers (542 and 633 nm), and one diode laser (405 nm).

Article Title: SIV Nef Proteins Recruit the AP-2 Complex to Antagonize Tetherin and Facilitate Virion Release
Article Snippet: Microscopy Cells stably expressing HA-tagged cpzTetherin or rhTetherin were seeded on 3.5-cm, glass-bottomed dishes coated with poly-L-Lysine (Mattek). .. Cells were transfected with 150 ng of an HIV-1 proviral plasmid (pBRHIV-1NL4-3ΔVpuΔNef) that was engineered to express various Nef proteins and 150 ng of an identical construct that expressed YFP embedded within the MA domain of Gag (pBRHIV-1NL4-3ΔVpuΔNef-MA(YFP)) using PEI (PolySciences). .. At 48 h post-transfection, cells were fixed with 4% paraformaldehyde and incubated with mouse anti-HA.11 monoclonal antibody (Covance) followed by anti-mouse IgG Alexafluor-594 conjugate (Molecular Probes).

Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling
Article Snippet: Necrostatin-1s was from Merck Millipore. .. Generation of HeLa-RIPK3 knockout variants To obtain HeLa-RIPK3-TRADDKO , HeLa-RIPK3-RIPK1KO , HeLa-RIPK3-FADDKO , and HeLa-RIPK3-casp8KO cells, HeLa-RIPK3 cells were transfected using polyethylenimine (PEI; Polysciences Inc., Warrington, USA) with mixtures of GeneArt CRISPR Nuclease (CD4 Reporter) vector plasmids (ThermoFisher Scientific) encoding three guide RNAs targeting the gene of interest and Cas9. ..

Article Title: Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif
Article Snippet: Statistical analyses were done with Prism 5 (GraphPad Software Inc.). .. Vif degradation 293T cells were transfected in triplicate with pVR1020-Vif-MYC or empty vector, at levels normalized by immunoblot, and pcDNA3.1 A3-HA, or empty vector, as indicated, using PEI (polyethyleneimine; Polysciences, Inc.). .. The following amounts of Vif expression construct were transfected for Fig. : HIV-1IIIB 50–200 ng; SIVmac239 50–200 ng; BIV 100–400 ng; FIV 50–200 ng; MVV 100–400 ng.

Article Title: Hypoxia-inducible factor 2α (HIF-2α) promotes colon cancer growth by potentiating Yes-associated protein 1 (YAP1) activity
Article Snippet: .. Enolase promoter luciferase reporter constructs Cyr61 or GTIIC activity reporter luciferase construct pGL4.47 (luc2P/SIE/Hygro) was co-transfected with YAP-1, HIF-2α-TM (HIF-2α triple mutant for protein stabilization), or empty vector (EV) into cells with polyethyleneimine (PEI; Polysciences Inc., Warrington, PA). .. Cells were treated with 100 n m Src family kinase inhibitor SU6656, AKT inhibitor MK2206, MAPK/ERK1/2 inhibitors GSK, or PD (PD325901) at 24 h after transfection.

Transfection:

Article Title: Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation
Article Snippet: .. Live cell imaging Plasmid (pEYFP-Nuc [a gift of Dr. T. Inoue], 0.25 µg) and pmCherry-SAMHD1 -HA (2 µg) were transfected into HEK293T cells using PEI Max (Polysciences) according to the manufacturer's protocol. .. For live cell imaging, HEK293T cells were transfected in 6-well coverslip glass-bottomed cell culture dishes (InVitro Scientific) when the cells were ∼80% confluent, and then visualized after 24 h using a Zeiss LSM510-Meta confocal imaging system equipped with four argon lasers (458-, 477-, 488-, and 514-nm lines), two HeNe lasers (542 and 633 nm), and one diode laser (405 nm).

Article Title: SIV Nef Proteins Recruit the AP-2 Complex to Antagonize Tetherin and Facilitate Virion Release
Article Snippet: Microscopy Cells stably expressing HA-tagged cpzTetherin or rhTetherin were seeded on 3.5-cm, glass-bottomed dishes coated with poly-L-Lysine (Mattek). .. Cells were transfected with 150 ng of an HIV-1 proviral plasmid (pBRHIV-1NL4-3ΔVpuΔNef) that was engineered to express various Nef proteins and 150 ng of an identical construct that expressed YFP embedded within the MA domain of Gag (pBRHIV-1NL4-3ΔVpuΔNef-MA(YFP)) using PEI (PolySciences). .. At 48 h post-transfection, cells were fixed with 4% paraformaldehyde and incubated with mouse anti-HA.11 monoclonal antibody (Covance) followed by anti-mouse IgG Alexafluor-594 conjugate (Molecular Probes).

Article Title: Protein Kinase D2 Assembles a Multiprotein Complex at the Trans-Golgi Network to Regulate Matrix Metalloproteinase Secretion *
Article Snippet: Experiments with ectopically expressed transgenes in HeLa cells were performed using HeLa Monster reagent (Mirus Bio, Madison, WI). .. HEK293T cells were transfected using PEI (Polysciences Inc., USA). .. N-terminal GFP-tagged and non-tagged pcDNA3 expression constructs for PKD1 and PKD2 have been described previously ( , ).

Article Title: Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome
Article Snippet: Fixed cells were mounted with Fluorogel Mounting Medium (EMS) and imaged with a Zeiss LSM700 confocal microscope. .. Immunoprecipitation and western blot GFP- and HA-tagged NARS2 constructs were co-expressed in HEK293T cells, after transfection using PEI reagent (Polysciences). .. Forty-eight hours after transfection, cells were harvested and homogenized with sonication in lysis buffer (50mM Tris HCl pH7.4, 100mM NaCl, 1% NP-40, 2mM Na3VO4) containing a protease inhibitor mixture (#P8340, Sigma).

Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling
Article Snippet: Necrostatin-1s was from Merck Millipore. .. Generation of HeLa-RIPK3 knockout variants To obtain HeLa-RIPK3-TRADDKO , HeLa-RIPK3-RIPK1KO , HeLa-RIPK3-FADDKO , and HeLa-RIPK3-casp8KO cells, HeLa-RIPK3 cells were transfected using polyethylenimine (PEI; Polysciences Inc., Warrington, USA) with mixtures of GeneArt CRISPR Nuclease (CD4 Reporter) vector plasmids (ThermoFisher Scientific) encoding three guide RNAs targeting the gene of interest and Cas9. ..

Article Title: Degradation of the cancer genomic DNA deaminase APOBEC3B by SIV Vif
Article Snippet: Statistical analyses were done with Prism 5 (GraphPad Software Inc.). .. Vif degradation 293T cells were transfected in triplicate with pVR1020-Vif-MYC or empty vector, at levels normalized by immunoblot, and pcDNA3.1 A3-HA, or empty vector, as indicated, using PEI (polyethyleneimine; Polysciences, Inc.). .. The following amounts of Vif expression construct were transfected for Fig. : HIV-1IIIB 50–200 ng; SIVmac239 50–200 ng; BIV 100–400 ng; FIV 50–200 ng; MVV 100–400 ng.

Article Title: Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2
Article Snippet: Eluates were buffer exchanged with PBS and concentrated using Amicon ultra filtration devices (Millipore Sigma) and stored at 4°C before use. .. Flow cytometry to test the binding of coronavirus RBD-Fc proteins to receptorsHEK293T cells were transfected with plasmids encoding ACE2 orthologs or human DPP4 using PEI 40K (Polysciences) according to manufacturer’s instructions. .. 48h post transfection, transfected cells were detached and incubated with 5 μg/ml RBD-Fc, and the interaction was detected with goat-anti-human-Ig-APC (Jackson ImmunoResearch Laboratories, Inc).

Construct:

Article Title: SIV Nef Proteins Recruit the AP-2 Complex to Antagonize Tetherin and Facilitate Virion Release
Article Snippet: Microscopy Cells stably expressing HA-tagged cpzTetherin or rhTetherin were seeded on 3.5-cm, glass-bottomed dishes coated with poly-L-Lysine (Mattek). .. Cells were transfected with 150 ng of an HIV-1 proviral plasmid (pBRHIV-1NL4-3ΔVpuΔNef) that was engineered to express various Nef proteins and 150 ng of an identical construct that expressed YFP embedded within the MA domain of Gag (pBRHIV-1NL4-3ΔVpuΔNef-MA(YFP)) using PEI (PolySciences). .. At 48 h post-transfection, cells were fixed with 4% paraformaldehyde and incubated with mouse anti-HA.11 monoclonal antibody (Covance) followed by anti-mouse IgG Alexafluor-594 conjugate (Molecular Probes).

Article Title: Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome
Article Snippet: Fixed cells were mounted with Fluorogel Mounting Medium (EMS) and imaged with a Zeiss LSM700 confocal microscope. .. Immunoprecipitation and western blot GFP- and HA-tagged NARS2 constructs were co-expressed in HEK293T cells, after transfection using PEI reagent (Polysciences). .. Forty-eight hours after transfection, cells were harvested and homogenized with sonication in lysis buffer (50mM Tris HCl pH7.4, 100mM NaCl, 1% NP-40, 2mM Na3VO4) containing a protease inhibitor mixture (#P8340, Sigma).

Article Title: Hypoxia-inducible factor 2α (HIF-2α) promotes colon cancer growth by potentiating Yes-associated protein 1 (YAP1) activity
Article Snippet: .. Enolase promoter luciferase reporter constructs Cyr61 or GTIIC activity reporter luciferase construct pGL4.47 (luc2P/SIE/Hygro) was co-transfected with YAP-1, HIF-2α-TM (HIF-2α triple mutant for protein stabilization), or empty vector (EV) into cells with polyethyleneimine (PEI; Polysciences Inc., Warrington, PA). .. Cells were treated with 100 n m Src family kinase inhibitor SU6656, AKT inhibitor MK2206, MAPK/ERK1/2 inhibitors GSK, or PD (PD325901) at 24 h after transfection.

Immunoprecipitation:

Article Title: Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome
Article Snippet: Fixed cells were mounted with Fluorogel Mounting Medium (EMS) and imaged with a Zeiss LSM700 confocal microscope. .. Immunoprecipitation and western blot GFP- and HA-tagged NARS2 constructs were co-expressed in HEK293T cells, after transfection using PEI reagent (Polysciences). .. Forty-eight hours after transfection, cells were harvested and homogenized with sonication in lysis buffer (50mM Tris HCl pH7.4, 100mM NaCl, 1% NP-40, 2mM Na3VO4) containing a protease inhibitor mixture (#P8340, Sigma).

Western Blot:

Article Title: Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase, Cause Nonsyndromic Deafness and Leigh Syndrome
Article Snippet: Fixed cells were mounted with Fluorogel Mounting Medium (EMS) and imaged with a Zeiss LSM700 confocal microscope. .. Immunoprecipitation and western blot GFP- and HA-tagged NARS2 constructs were co-expressed in HEK293T cells, after transfection using PEI reagent (Polysciences). .. Forty-eight hours after transfection, cells were harvested and homogenized with sonication in lysis buffer (50mM Tris HCl pH7.4, 100mM NaCl, 1% NP-40, 2mM Na3VO4) containing a protease inhibitor mixture (#P8340, Sigma).

Knock-Out:

Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling
Article Snippet: Necrostatin-1s was from Merck Millipore. .. Generation of HeLa-RIPK3 knockout variants To obtain HeLa-RIPK3-TRADDKO , HeLa-RIPK3-RIPK1KO , HeLa-RIPK3-FADDKO , and HeLa-RIPK3-casp8KO cells, HeLa-RIPK3 cells were transfected using polyethylenimine (PEI; Polysciences Inc., Warrington, USA) with mixtures of GeneArt CRISPR Nuclease (CD4 Reporter) vector plasmids (ThermoFisher Scientific) encoding three guide RNAs targeting the gene of interest and Cas9. ..

CRISPR:

Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling
Article Snippet: Necrostatin-1s was from Merck Millipore. .. Generation of HeLa-RIPK3 knockout variants To obtain HeLa-RIPK3-TRADDKO , HeLa-RIPK3-RIPK1KO , HeLa-RIPK3-FADDKO , and HeLa-RIPK3-casp8KO cells, HeLa-RIPK3 cells were transfected using polyethylenimine (PEI; Polysciences Inc., Warrington, USA) with mixtures of GeneArt CRISPR Nuclease (CD4 Reporter) vector plasmids (ThermoFisher Scientific) encoding three guide RNAs targeting the gene of interest and Cas9. ..

Luciferase:

Article Title: Hypoxia-inducible factor 2α (HIF-2α) promotes colon cancer growth by potentiating Yes-associated protein 1 (YAP1) activity
Article Snippet: .. Enolase promoter luciferase reporter constructs Cyr61 or GTIIC activity reporter luciferase construct pGL4.47 (luc2P/SIE/Hygro) was co-transfected with YAP-1, HIF-2α-TM (HIF-2α triple mutant for protein stabilization), or empty vector (EV) into cells with polyethyleneimine (PEI; Polysciences Inc., Warrington, PA). .. Cells were treated with 100 n m Src family kinase inhibitor SU6656, AKT inhibitor MK2206, MAPK/ERK1/2 inhibitors GSK, or PD (PD325901) at 24 h after transfection.

Activity Assay:

Article Title: Hypoxia-inducible factor 2α (HIF-2α) promotes colon cancer growth by potentiating Yes-associated protein 1 (YAP1) activity
Article Snippet: .. Enolase promoter luciferase reporter constructs Cyr61 or GTIIC activity reporter luciferase construct pGL4.47 (luc2P/SIE/Hygro) was co-transfected with YAP-1, HIF-2α-TM (HIF-2α triple mutant for protein stabilization), or empty vector (EV) into cells with polyethyleneimine (PEI; Polysciences Inc., Warrington, PA). .. Cells were treated with 100 n m Src family kinase inhibitor SU6656, AKT inhibitor MK2206, MAPK/ERK1/2 inhibitors GSK, or PD (PD325901) at 24 h after transfection.

Mutagenesis:

Article Title: Hypoxia-inducible factor 2α (HIF-2α) promotes colon cancer growth by potentiating Yes-associated protein 1 (YAP1) activity
Article Snippet: .. Enolase promoter luciferase reporter constructs Cyr61 or GTIIC activity reporter luciferase construct pGL4.47 (luc2P/SIE/Hygro) was co-transfected with YAP-1, HIF-2α-TM (HIF-2α triple mutant for protein stabilization), or empty vector (EV) into cells with polyethyleneimine (PEI; Polysciences Inc., Warrington, PA). .. Cells were treated with 100 n m Src family kinase inhibitor SU6656, AKT inhibitor MK2206, MAPK/ERK1/2 inhibitors GSK, or PD (PD325901) at 24 h after transfection.

Flow Cytometry:

Article Title: Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2
Article Snippet: Eluates were buffer exchanged with PBS and concentrated using Amicon ultra filtration devices (Millipore Sigma) and stored at 4°C before use. .. Flow cytometry to test the binding of coronavirus RBD-Fc proteins to receptorsHEK293T cells were transfected with plasmids encoding ACE2 orthologs or human DPP4 using PEI 40K (Polysciences) according to manufacturer’s instructions. .. 48h post transfection, transfected cells were detached and incubated with 5 μg/ml RBD-Fc, and the interaction was detected with goat-anti-human-Ig-APC (Jackson ImmunoResearch Laboratories, Inc).

Binding Assay:

Article Title: Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2
Article Snippet: Eluates were buffer exchanged with PBS and concentrated using Amicon ultra filtration devices (Millipore Sigma) and stored at 4°C before use. .. Flow cytometry to test the binding of coronavirus RBD-Fc proteins to receptorsHEK293T cells were transfected with plasmids encoding ACE2 orthologs or human DPP4 using PEI 40K (Polysciences) according to manufacturer’s instructions. .. 48h post transfection, transfected cells were detached and incubated with 5 μg/ml RBD-Fc, and the interaction was detected with goat-anti-human-Ig-APC (Jackson ImmunoResearch Laboratories, Inc).

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Polysciences inc pei max
    WT SAMHD1 relocates cytoplasm-localized mutants of SAMHD1K11A to the nucleus and facilitates their Vpx-induced degradation. (A)–(C) Wild-type SAMHD1-HA partially translocated <t>pmCherry-SAMHD1</t> K11A to the nucleus. pmCherry-SAMHD1-HA WT and/or K11A with pEYFP-Nuc were co-transfected into HEK293T cells using <t>PEI</t> Max. Live cells were imaged at 24 h post-transfection. (D) Interaction between wild-type SAMHD1 and K11A. pSAMHD1 WT-Flag was co-transfected with pmCherry-SAMHD1-HA WT or K11A. Transfected cells were harvested after 48 h and incubated in lysis buffer for 30 min. Cell lysates were added to an anti-HA affinity matrix (Roche). Eluted protein samples were detected by immunoblotting with anti-HA and anti-Flag antibody to detect mCherry-SAMHD1 and SAMHD1-Flag, respectively. (E) Immunoblotting demonstrates that pmCherry-SAMHD1 K11A became sensitive to Vpx-mediated degradation when co-expressed with SAMHD1 WT. HEK293T cells were co-transfected with HA-Vpx and pmCherrySAMHD1 K11A and/or pSAMHD1-Flag wild-type. Cell extracts were harvested 48 h later and analyzed by SDS-PAGE, followed by immunoblotting to detect SAMHD1-HA and HA-Vpx. β-actin was used as the loading control.
    Pei Max, supplied by Polysciences inc, 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/pei max/product/Polysciences inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pei max - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    86
    Polysciences inc pei
    In vivo uptake of polyplexes by DCs in the draining lymph nodes 24 h after s.c. injection in mice. Polyplexes were prepared from <t>PEG-</t> b -PAEM 75 or <t>PEI</t> with anionic fluorescently-labeled dextran (Dex). For comparison, Dex alone and 100-nm fluorescent polystyrene
    Pei, supplied by Polysciences inc, 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/pei/product/Polysciences inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pei - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    86
    Polysciences inc high molecular weight pei
    LLO activity of <t>LLO-s-s-PEI</t> monitored by in vitro red blood cell hemolysis assay. <t>LLO-s-s-PEI25</t> was assayed for its ability to lyse red blood cells (RBCs) by monitoring the change in scattering of 590 nm light by 2×10 8 RBCs/ml in a Spex Fluoromax-2
    High Molecular Weight Pei, supplied by Polysciences inc, 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/high molecular weight pei/product/Polysciences inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    high molecular weight pei - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    86
    Polysciences inc linear pei
    Effect of DNA to <t>PEI</t> ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 <t>kDa</t> PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.
    Linear Pei, supplied by Polysciences inc, 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/linear pei/product/Polysciences inc
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    linear pei - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    Image Search Results


    WT SAMHD1 relocates cytoplasm-localized mutants of SAMHD1K11A to the nucleus and facilitates their Vpx-induced degradation. (A)–(C) Wild-type SAMHD1-HA partially translocated pmCherry-SAMHD1 K11A to the nucleus. pmCherry-SAMHD1-HA WT and/or K11A with pEYFP-Nuc were co-transfected into HEK293T cells using PEI Max. Live cells were imaged at 24 h post-transfection. (D) Interaction between wild-type SAMHD1 and K11A. pSAMHD1 WT-Flag was co-transfected with pmCherry-SAMHD1-HA WT or K11A. Transfected cells were harvested after 48 h and incubated in lysis buffer for 30 min. Cell lysates were added to an anti-HA affinity matrix (Roche). Eluted protein samples were detected by immunoblotting with anti-HA and anti-Flag antibody to detect mCherry-SAMHD1 and SAMHD1-Flag, respectively. (E) Immunoblotting demonstrates that pmCherry-SAMHD1 K11A became sensitive to Vpx-mediated degradation when co-expressed with SAMHD1 WT. HEK293T cells were co-transfected with HA-Vpx and pmCherrySAMHD1 K11A and/or pSAMHD1-Flag wild-type. Cell extracts were harvested 48 h later and analyzed by SDS-PAGE, followed by immunoblotting to detect SAMHD1-HA and HA-Vpx. β-actin was used as the loading control.

    Journal: PLoS ONE

    Article Title: Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation

    doi: 10.1371/journal.pone.0066201

    Figure Lengend Snippet: WT SAMHD1 relocates cytoplasm-localized mutants of SAMHD1K11A to the nucleus and facilitates their Vpx-induced degradation. (A)–(C) Wild-type SAMHD1-HA partially translocated pmCherry-SAMHD1 K11A to the nucleus. pmCherry-SAMHD1-HA WT and/or K11A with pEYFP-Nuc were co-transfected into HEK293T cells using PEI Max. Live cells were imaged at 24 h post-transfection. (D) Interaction between wild-type SAMHD1 and K11A. pSAMHD1 WT-Flag was co-transfected with pmCherry-SAMHD1-HA WT or K11A. Transfected cells were harvested after 48 h and incubated in lysis buffer for 30 min. Cell lysates were added to an anti-HA affinity matrix (Roche). Eluted protein samples were detected by immunoblotting with anti-HA and anti-Flag antibody to detect mCherry-SAMHD1 and SAMHD1-Flag, respectively. (E) Immunoblotting demonstrates that pmCherry-SAMHD1 K11A became sensitive to Vpx-mediated degradation when co-expressed with SAMHD1 WT. HEK293T cells were co-transfected with HA-Vpx and pmCherrySAMHD1 K11A and/or pSAMHD1-Flag wild-type. Cell extracts were harvested 48 h later and analyzed by SDS-PAGE, followed by immunoblotting to detect SAMHD1-HA and HA-Vpx. β-actin was used as the loading control.

    Article Snippet: Live cell imaging Plasmid (pEYFP-Nuc [a gift of Dr. T. Inoue], 0.25 µg) and pmCherry-SAMHD1 -HA (2 µg) were transfected into HEK293T cells using PEI Max (Polysciences) according to the manufacturer's protocol.

    Techniques: Transfection, Incubation, Lysis, SDS Page

    Cellular localization of mCherry-tagged SAMHD1 and SAMHD1 mutant proteins using live cell imaging. (A)–(F) Plasmid pEYFP-Nuc (Clontech) and pmCherry-SAMHD1 WT or indicated variants were co-transfected into HEK293T cells using PEI Max (Polysciences). For live cell imaging, HEK293T cells were transfected in 6-well coverslip glass-bottomed cell culture dishes (InVitro Scientific) when the cells were ∼80% confluent, and then visualized after 24 h using a Zeiss LSM510-Meta confocal imaging system equipped with four argon lasers (458-, 477-, 488-, and 514-nm lines), two HeNe lasers (542 and 633 nm), and one diode laser (405 nm). All images were acquired from a 100X objective, and image analysis and manipulation were performed using Zen 2009 software.

    Journal: PLoS ONE

    Article Title: Identification of Critical Regions in Human SAMHD1 Required for Nuclear Localization and Vpx-Mediated Degradation

    doi: 10.1371/journal.pone.0066201

    Figure Lengend Snippet: Cellular localization of mCherry-tagged SAMHD1 and SAMHD1 mutant proteins using live cell imaging. (A)–(F) Plasmid pEYFP-Nuc (Clontech) and pmCherry-SAMHD1 WT or indicated variants were co-transfected into HEK293T cells using PEI Max (Polysciences). For live cell imaging, HEK293T cells were transfected in 6-well coverslip glass-bottomed cell culture dishes (InVitro Scientific) when the cells were ∼80% confluent, and then visualized after 24 h using a Zeiss LSM510-Meta confocal imaging system equipped with four argon lasers (458-, 477-, 488-, and 514-nm lines), two HeNe lasers (542 and 633 nm), and one diode laser (405 nm). All images were acquired from a 100X objective, and image analysis and manipulation were performed using Zen 2009 software.

    Article Snippet: Live cell imaging Plasmid (pEYFP-Nuc [a gift of Dr. T. Inoue], 0.25 µg) and pmCherry-SAMHD1 -HA (2 µg) were transfected into HEK293T cells using PEI Max (Polysciences) according to the manufacturer's protocol.

    Techniques: Mutagenesis, Live Cell Imaging, Plasmid Preparation, Transfection, Cell Culture, Imaging, Software

    In vivo uptake of polyplexes by DCs in the draining lymph nodes 24 h after s.c. injection in mice. Polyplexes were prepared from PEG- b -PAEM 75 or PEI with anionic fluorescently-labeled dextran (Dex). For comparison, Dex alone and 100-nm fluorescent polystyrene

    Journal: Journal of controlled release : official journal of the Controlled Release Society

    Article Title: Well-defined Block Copolymers for Gene Delivery to Dendritic Cells: Probing the Effect of Polycation Chain-length

    doi: 10.1016/j.jconrel.2009.10.021

    Figure Lengend Snippet: In vivo uptake of polyplexes by DCs in the draining lymph nodes 24 h after s.c. injection in mice. Polyplexes were prepared from PEG- b -PAEM 75 or PEI with anionic fluorescently-labeled dextran (Dex). For comparison, Dex alone and 100-nm fluorescent polystyrene

    Article Snippet: Monomethoxy-PEG (average M n of 2,000) and PEI (branched, 25 kDa) were purchased from Polysciences.

    Techniques: In Vivo, Injection, Mouse Assay, Labeling

    LLO activity of LLO-s-s-PEI monitored by in vitro red blood cell hemolysis assay. LLO-s-s-PEI25 was assayed for its ability to lyse red blood cells (RBCs) by monitoring the change in scattering of 590 nm light by 2×10 8 RBCs/ml in a Spex Fluoromax-2

    Journal:

    Article Title: Enhanced gene delivery using disulfide-crosslinked low molecular weight polyethylenimine with listeriolysin o-polyethylenimine disulfide conjugate

    doi: 10.1016/j.jconrel.2008.07.007

    Figure Lengend Snippet: LLO activity of LLO-s-s-PEI monitored by in vitro red blood cell hemolysis assay. LLO-s-s-PEI25 was assayed for its ability to lyse red blood cells (RBCs) by monitoring the change in scattering of 590 nm light by 2×10 8 RBCs/ml in a Spex Fluoromax-2

    Article Snippet: Low molecular weight PEI (branched, average molecular weight 1.8 kDa: PEI1.8) and high molecular weight PEI (branched, average molecular weight 25 kDa: PEI25) were purchased from Polysciences Inc (Warrington, PA).

    Techniques: Activity Assay, In Vitro, Hemolysis Assay

    Transfection with three different forms of PEIs (different molecular weights and different crosslinking: s-s PEI1.8; PEI1.8; PEI25) (a) The effect of disulfide-crosslinking of PEI1.8 on luciferase gene expression in HEK 293 cells at different PEI/pDNA

    Journal:

    Article Title: Enhanced gene delivery using disulfide-crosslinked low molecular weight polyethylenimine with listeriolysin o-polyethylenimine disulfide conjugate

    doi: 10.1016/j.jconrel.2008.07.007

    Figure Lengend Snippet: Transfection with three different forms of PEIs (different molecular weights and different crosslinking: s-s PEI1.8; PEI1.8; PEI25) (a) The effect of disulfide-crosslinking of PEI1.8 on luciferase gene expression in HEK 293 cells at different PEI/pDNA

    Article Snippet: Low molecular weight PEI (branched, average molecular weight 1.8 kDa: PEI1.8) and high molecular weight PEI (branched, average molecular weight 25 kDa: PEI25) were purchased from Polysciences Inc (Warrington, PA).

    Techniques: Transfection, Luciferase, Expressing

    The effect of increasing amounts of LLO-s-s-PEI25, incorporated in the PEI/pDNA condensates, on luciferase gene expression in HEK 293 cells. The amount of LLO-s-s-PEI25 incorporated in the PEI/pDNA complex (expressed as w/w % of LLO-s-s-PEI25 relative

    Journal:

    Article Title: Enhanced gene delivery using disulfide-crosslinked low molecular weight polyethylenimine with listeriolysin o-polyethylenimine disulfide conjugate

    doi: 10.1016/j.jconrel.2008.07.007

    Figure Lengend Snippet: The effect of increasing amounts of LLO-s-s-PEI25, incorporated in the PEI/pDNA condensates, on luciferase gene expression in HEK 293 cells. The amount of LLO-s-s-PEI25 incorporated in the PEI/pDNA complex (expressed as w/w % of LLO-s-s-PEI25 relative

    Article Snippet: Low molecular weight PEI (branched, average molecular weight 1.8 kDa: PEI1.8) and high molecular weight PEI (branched, average molecular weight 25 kDa: PEI25) were purchased from Polysciences Inc (Warrington, PA).

    Techniques: Luciferase, Expressing

    Effect of DNA to PEI ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.

    Journal: Nucleic Acids Research

    Article Title: High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells

    doi:

    Figure Lengend Snippet: Effect of DNA to PEI ratio on transfection efficiency. 293E cells were transfected with linear ( A ) or branched ( B ) 25 kDa PEI at various DNA (pEGFP plasmid) concentrations as described in Materials and Methods. DNA concentrations (µg ml –1 ) used were: 0.25 (circles), 0.50 (squares), 1.0 (closed diamonds), 1.5 (triangles) and 2.0 (open diamonds). Transfection efficiencies were determined by flow cytometry analysis 72 hpt.

    Article Snippet: A 25 kDa branched PEI was obtained from Aldrich (Milwaukee, WI) and 25 kDa linear PEI from Polysciences (Warrington, PA).

    Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Cytometry