superfect  (Qiagen)

 
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    Name:
    SuperFect Transfection Reagent
    Description:
    For transfection of a broad range of eukaryotic cell lines with DNA Kit contents Qiagen SuperFect Transfection Reagent 1 2mL For 40 Transfections in 160mm Dishes or 640 Transfections in 12 well Plates Eukaryotic Cell Type Ideal for Transient and Stable Transfection of a Broad Range of Cell Lines Including 293 B16 BHK 21 COS 1 and CHO SuperFect Reagent is Suited for Studies on Gene Expression and Function Drug Discovery Development Studies Activated dendrimer Technology Excellent Reproducibility and Low Cytotoxicity Benefits Suitable for a broad range of cell lines Transfection can be performed in the presence of serum Activated dendrimers ensure reproducibility
    Catalog Number:
    301305
    Price:
    312
    Category:
    SuperFect Transfection Reagent
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    Structured Review

    Qiagen superfect
    SuperFect Transfection Reagent
    For transfection of a broad range of eukaryotic cell lines with DNA Kit contents Qiagen SuperFect Transfection Reagent 1 2mL For 40 Transfections in 160mm Dishes or 640 Transfections in 12 well Plates Eukaryotic Cell Type Ideal for Transient and Stable Transfection of a Broad Range of Cell Lines Including 293 B16 BHK 21 COS 1 and CHO SuperFect Reagent is Suited for Studies on Gene Expression and Function Drug Discovery Development Studies Activated dendrimer Technology Excellent Reproducibility and Low Cytotoxicity Benefits Suitable for a broad range of cell lines Transfection can be performed in the presence of serum Activated dendrimers ensure reproducibility
    https://www.bioz.com/result/superfect/product/Qiagen
    Average 99 stars, based on 1366 article reviews
    Price from $9.99 to $1999.99
    superfect - by Bioz Stars, 2020-11
    99/100 stars

    Images

    1) Product Images from "NF-?B Controls Cell Growth and Differentiation through Transcriptional Regulation of Cyclin D1"

    Article Title: NF-?B Controls Cell Growth and Differentiation through Transcriptional Regulation of Cyclin D1

    Journal: Molecular and Cellular Biology

    doi:

    NF-κB inhibits MyoD-induced myogenesis in 10T1/2 fibroblasts. Cells were maintained in growth medium containing 15% FBS and differentiated in DM. The cells were seeded in triplicate overnight in 6-cm dishes, and the following day, cotransfections were performed with Superfect (Qiagen). DNA consisted of 1 μg of a troponin-I-Luc reporter plasmid (TnI-Luc) (A) or 1 μg of the 4RTK-Luc plasmid (B), along with 0.25 μg of an expression plasmid for MyoD alone or in combination with 0.5 μg of expression plasmids for either the activated form of oncogenic ras (H- ras V-12), p65, p50, or 1 μg of IκBαSR. DNA was standardized to 2.5 μg by the addition of Bluescript plasmid (Stratagene). Cells were maintained in growth medium for 24 h following transfections and then switched to DM for 48 h, at which time cell extracts were prepared and relative light units (RLU) were determined, by normalizing values to total protein. (C) For immunofluorescence analysis, 10T1/2 cells were seeded overnight and the next day similar transfections were performed as described above, except that one-third of the amount of DNA was used. At 24 h following transfections, the cells were switched to DM for 72 h, at which time the cells were fixed and probed for the myosin heavy chain. To score for the number of myotubes formed, cells expressing myosin were counted and averaged from a minimum of 10 randomly selected fields.
    Figure Legend Snippet: NF-κB inhibits MyoD-induced myogenesis in 10T1/2 fibroblasts. Cells were maintained in growth medium containing 15% FBS and differentiated in DM. The cells were seeded in triplicate overnight in 6-cm dishes, and the following day, cotransfections were performed with Superfect (Qiagen). DNA consisted of 1 μg of a troponin-I-Luc reporter plasmid (TnI-Luc) (A) or 1 μg of the 4RTK-Luc plasmid (B), along with 0.25 μg of an expression plasmid for MyoD alone or in combination with 0.5 μg of expression plasmids for either the activated form of oncogenic ras (H- ras V-12), p65, p50, or 1 μg of IκBαSR. DNA was standardized to 2.5 μg by the addition of Bluescript plasmid (Stratagene). Cells were maintained in growth medium for 24 h following transfections and then switched to DM for 48 h, at which time cell extracts were prepared and relative light units (RLU) were determined, by normalizing values to total protein. (C) For immunofluorescence analysis, 10T1/2 cells were seeded overnight and the next day similar transfections were performed as described above, except that one-third of the amount of DNA was used. At 24 h following transfections, the cells were switched to DM for 72 h, at which time the cells were fixed and probed for the myosin heavy chain. To score for the number of myotubes formed, cells expressing myosin were counted and averaged from a minimum of 10 randomly selected fields.

    Techniques Used: Plasmid Preparation, Expressing, Transfection, Immunofluorescence

    2) Product Images from "Oxalate-inducible AMBP gene and its regulatory mechanism in renal tubular epithelial cells"

    Article Title: Oxalate-inducible AMBP gene and its regulatory mechanism in renal tubular epithelial cells

    Journal: Biochemical Journal

    doi: 10.1042/BJ20041465

    Sequestering of HNF-4 by ApH4ON decreases endogenous A1M synthesis in LLC-PK1 cells Semi-confluent LLC-PK1 cells were transfected with double-stranded oligonucleotide ApH4ON or OctON (a non-specific oligonucleotide) using SuperFect™ reagent following the manufacturer's instructions (Qiagen). Cells transfected with ApH4ON showed substantially lower endogenous production of A1M, whereas those transfected with OctON did not. ( A ) A1M protein in cells treated with vehicle alone and cells transfected with ApH4ON and OctON on exposure to oxalate for 3 h. ( B ) Graphical representation of results shown in ( A ). Results represent two-way ANOVA with means±S.E.M. ( n =3). * P
    Figure Legend Snippet: Sequestering of HNF-4 by ApH4ON decreases endogenous A1M synthesis in LLC-PK1 cells Semi-confluent LLC-PK1 cells were transfected with double-stranded oligonucleotide ApH4ON or OctON (a non-specific oligonucleotide) using SuperFect™ reagent following the manufacturer's instructions (Qiagen). Cells transfected with ApH4ON showed substantially lower endogenous production of A1M, whereas those transfected with OctON did not. ( A ) A1M protein in cells treated with vehicle alone and cells transfected with ApH4ON and OctON on exposure to oxalate for 3 h. ( B ) Graphical representation of results shown in ( A ). Results represent two-way ANOVA with means±S.E.M. ( n =3). * P

    Techniques Used: Transfection

    3) Product Images from "Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells"

    Article Title: Peptide-mediated RNA delivery: a novel approach for enhanced transfection of primary and post-mitotic cells

    Journal: Nucleic Acids Research

    doi:

    Lipoplexes or polyplexes/mRNA-mediated transfection of B16-F10 cells. cap- luc -A 30 (1 µg) condensed by cationic lipids (DOTAP N/P = 1.8, DOGS six charge equivalent) or cationic polymers (PEI 25 kDa N/P = 10, PEI 22 kDa N/P = 5, PLL 54 kDa N/P = 2 and Superfect N/P = 8.5) were delivered to B16-F10 cells and luciferase gene expression evaluated 6 h post-transfection. The efficiency of transfection was compared to the pGL3 luc /DOTAP lipoplexes (luciferase expression measured 24 h post-transfection). Each experiment was performed in triplicate. nd , not detected.
    Figure Legend Snippet: Lipoplexes or polyplexes/mRNA-mediated transfection of B16-F10 cells. cap- luc -A 30 (1 µg) condensed by cationic lipids (DOTAP N/P = 1.8, DOGS six charge equivalent) or cationic polymers (PEI 25 kDa N/P = 10, PEI 22 kDa N/P = 5, PLL 54 kDa N/P = 2 and Superfect N/P = 8.5) were delivered to B16-F10 cells and luciferase gene expression evaluated 6 h post-transfection. The efficiency of transfection was compared to the pGL3 luc /DOTAP lipoplexes (luciferase expression measured 24 h post-transfection). Each experiment was performed in triplicate. nd , not detected.

    Techniques Used: Transfection, Luciferase, Expressing

    4) Product Images from "Increase of androgen-induced cell death and androgen receptor transactivation by BRCA1 in prostate cancer cells"

    Article Title: Increase of androgen-induced cell death and androgen receptor transactivation by BRCA1 in prostate cancer cells

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

    doi:

    The interaction between BRCA1 and AR. ( A ) Mapping the domains of BRCA1 that are responsible for AR interaction. Six recombinant GST–BRCA1 fusion proteins, fragments #1, #2, #3, #4, #5, and #6, were generated in Escherichia coli as described. BRCA1 residues are marked relative to the translation initiation site. The Coomassie blue-stained SDS polyacrylamide gel, showing the relative abundance of each fusion protein, was used in the GST pull-down assay as described. The 110-kDa protein bound to GST–BRCA1 #4 and #6 is a product of 35 S-methionine-labeled full-length AR. ( B ) Mapping the domain of AR that is required for BRCA1 interaction. 20 μl of in vitro translated 35 S-methionine-labeled AR N (from amino acids 36–553) and AR DBD-LBD (from amino acids 553–918) protein was used to perform the pull-down assay. The results indicated that GST–BRCA1 fragment #4 (amino acids 758-1064) can interact both with the N-terminal and DBD-LBD of AR. In contrast, GST–BRCA1 #6 (amino acids 1314–1863) can associate with only the DBD- LBD of AR. Our data indicate that there are two contact pockets between BRCA1 and AR. ( C ) The interaction between AR and BRCA1 by mammalian two-hybrid assay. PC-3 cells in 35-mm dishes were transiently cotransfected with 0.5 μg of reporter plasmid pG5-LUC, and 0.75 μg of Gal4DBD fused BRCA1 constructs, amino acids 1–304, amino acids 231-1314, amino acids 1560–1863, with or without 0.75 μg of VP16 fused AR (VP16-AR) construct for 2 h by SuperFect . 1 nM DHT was added for another 24 h, and then the cells were harvested for LUC assay. Arrows indicate VP16-fused SV40 large T antigen and ARA70 were applied here to assure the interaction specificity between BRCA1 and AR. The results indicate that BRCA1 amino acids 231-1314 and amino acids 1560–1863 are responsible for AR interaction; these results are consistent with the results from GST pull-down assay.
    Figure Legend Snippet: The interaction between BRCA1 and AR. ( A ) Mapping the domains of BRCA1 that are responsible for AR interaction. Six recombinant GST–BRCA1 fusion proteins, fragments #1, #2, #3, #4, #5, and #6, were generated in Escherichia coli as described. BRCA1 residues are marked relative to the translation initiation site. The Coomassie blue-stained SDS polyacrylamide gel, showing the relative abundance of each fusion protein, was used in the GST pull-down assay as described. The 110-kDa protein bound to GST–BRCA1 #4 and #6 is a product of 35 S-methionine-labeled full-length AR. ( B ) Mapping the domain of AR that is required for BRCA1 interaction. 20 μl of in vitro translated 35 S-methionine-labeled AR N (from amino acids 36–553) and AR DBD-LBD (from amino acids 553–918) protein was used to perform the pull-down assay. The results indicated that GST–BRCA1 fragment #4 (amino acids 758-1064) can interact both with the N-terminal and DBD-LBD of AR. In contrast, GST–BRCA1 #6 (amino acids 1314–1863) can associate with only the DBD- LBD of AR. Our data indicate that there are two contact pockets between BRCA1 and AR. ( C ) The interaction between AR and BRCA1 by mammalian two-hybrid assay. PC-3 cells in 35-mm dishes were transiently cotransfected with 0.5 μg of reporter plasmid pG5-LUC, and 0.75 μg of Gal4DBD fused BRCA1 constructs, amino acids 1–304, amino acids 231-1314, amino acids 1560–1863, with or without 0.75 μg of VP16 fused AR (VP16-AR) construct for 2 h by SuperFect . 1 nM DHT was added for another 24 h, and then the cells were harvested for LUC assay. Arrows indicate VP16-fused SV40 large T antigen and ARA70 were applied here to assure the interaction specificity between BRCA1 and AR. The results indicate that BRCA1 amino acids 231-1314 and amino acids 1560–1863 are responsible for AR interaction; these results are consistent with the results from GST pull-down assay.

    Techniques Used: Recombinant, Generated, Staining, Pull Down Assay, Labeling, In Vitro, Two Hybrid Assay, Plasmid Preparation, Construct

    Potentiation of the AR transactivation by BRCA1. ( A ). The total plasmid amount was adjusted with pSG5, pCR3, or pCMV parent vector to 11 μg for each 60-mm transfection by calcium phosphate precipitation method. ( B ) BRCA1 can potentiate the AR transactivation in PC-3 cell. Cells were transfected as mentioned above. ( C ) BRCA1 can potentiate the AR transactivation in LNCaP cells in the presence of androgen without changing the expression of AR. 0.5 μg of PSA-LUC and 1.0 μg of pCR3 or pCR3-BRCA1 were transfected into LNCaP cells in 35-mm dish for 2 h by SuperFect . Cells were then treated with 1 nM DHT for an additional 24 h and harvested for LUC assay. The relative LUC activity was normalized against Renilla LUC activity (Promega). Data represent an average of three independent experiments. Duplicate LNCaP cells were harvested, and 60 μ g of whole-cell extract was assayed with Western blotting for the detection of AR protein. The ectopically expressed BRCA1 cannot affect the expression of endogenous AR in LNCaP cells. ( D ) AR coregulators could cooperate with BRCA1 to synergistically enhance the AR transactivation. DU145 cells were cotransfected with 3 μg of MMTV-CAT, 1 μg of pSG5-AR, and 3 μg of alone or together with 3 μg CBP, ARA70N, ARA55, or BRCA1, in the absence or presence of 1 nM DHT. The error bars represent the mean ± SD of four independent experiments. ( E ) BRCA1 can potentiate the AR transactivation in MCF-7 and T47D cells. 0.5 μg of PSA-LUC reporter plasmid or 1.0 μg of pCR3-BRCA1 were transfected into T47D and MCF-7 cells. Cells were treated with 1 nM DHT after transfection as mentioned above.
    Figure Legend Snippet: Potentiation of the AR transactivation by BRCA1. ( A ). The total plasmid amount was adjusted with pSG5, pCR3, or pCMV parent vector to 11 μg for each 60-mm transfection by calcium phosphate precipitation method. ( B ) BRCA1 can potentiate the AR transactivation in PC-3 cell. Cells were transfected as mentioned above. ( C ) BRCA1 can potentiate the AR transactivation in LNCaP cells in the presence of androgen without changing the expression of AR. 0.5 μg of PSA-LUC and 1.0 μg of pCR3 or pCR3-BRCA1 were transfected into LNCaP cells in 35-mm dish for 2 h by SuperFect . Cells were then treated with 1 nM DHT for an additional 24 h and harvested for LUC assay. The relative LUC activity was normalized against Renilla LUC activity (Promega). Data represent an average of three independent experiments. Duplicate LNCaP cells were harvested, and 60 μ g of whole-cell extract was assayed with Western blotting for the detection of AR protein. The ectopically expressed BRCA1 cannot affect the expression of endogenous AR in LNCaP cells. ( D ) AR coregulators could cooperate with BRCA1 to synergistically enhance the AR transactivation. DU145 cells were cotransfected with 3 μg of MMTV-CAT, 1 μg of pSG5-AR, and 3 μg of alone or together with 3 μg CBP, ARA70N, ARA55, or BRCA1, in the absence or presence of 1 nM DHT. The error bars represent the mean ± SD of four independent experiments. ( E ) BRCA1 can potentiate the AR transactivation in MCF-7 and T47D cells. 0.5 μg of PSA-LUC reporter plasmid or 1.0 μg of pCR3-BRCA1 were transfected into T47D and MCF-7 cells. Cells were treated with 1 nM DHT after transfection as mentioned above.

    Techniques Used: Plasmid Preparation, Transfection, Expressing, Activity Assay, Western Blot

    The AR and BRCA1 could cooperatively regulate the promoter activity and protein expression of p21 (WAF1/CIP1) and stimulate the cell death in PC-3(AR2). ( A ) The coexpression of AR and BRCA1 cooperatively induces the −291 and −2326 p21 (WAF1/CIP1) promoter but not the basal promoter activity. In each transfection, 0.5 μg of reporter gene, 0.5 μg of AR, with or without 1 μg of BRCA1, were cotransfected into PC-3 cells. After 2 h of transfection, the medium was changed, and 10 nM DHT was added for another 30 h. ( B ) The endogenous p21 (WAF1/CIP1) expression was induced by DHT-AR and BRCA1 in MCF-7 cells. MCF-7 cells were seeded on 60-mm wells and cotransfected with 2 μg of AR with or without 4 μg of BRCA1 by SuperFect . After 2 h of transfection, the medium was changed, and 10 nM DHT was applied for another 30 h. In each experiment, 60 μg of whole-cell extract was applied for the Western blotting. ( C ) The p21 (WAF1/CIP1) protein is enhanced by DHT/AR and inhibited by hydroxyflutamide (HF) in PC-3(AR2) cells. The expression of protein p21 (WAF1/CIP1) can be induced by DHT, and this induction can be inhibited by 1 μM HF (lane 2 vs. lane 3). ( D ) The p21 (WAF1/CIP1) protein is enhanced by DHT/AR and BRCA1 in PC-3(AR2). PC-3(AR2) cells were cotransfected with or without 4 μg of BRCA1. After 2 h of transfection, the medium was changed, and 10 nM DHT was applied for another 30 h. In each experiment, 60 μg of whole-cell extract was applied for the Western blotting. ( E ) Cell growth is regulated by DHT/AR and BRCA1 in PC-3(AR2) cells. Duplicate PC-3(AR2) cells (as in D ) were applied to MTT assay for the relative cell number determination. ( F ) Dead cells were indicated as loss of cell membrane integrity assayed by PI inclusion. PC-3(AR2) cells were transfected with 4 μg of BRCA1. After 2 h of transfection, the medium was changed, and 10 nM DHT or vehicle was applied for another 4 days. The medium was changed on day 2. Attached cells were trypsinized and collected with floating cells, stained with 20 μg/ml PI on day 4. After 10 min of staining, the PI-positive cells were then counted under fluorescent microscope.
    Figure Legend Snippet: The AR and BRCA1 could cooperatively regulate the promoter activity and protein expression of p21 (WAF1/CIP1) and stimulate the cell death in PC-3(AR2). ( A ) The coexpression of AR and BRCA1 cooperatively induces the −291 and −2326 p21 (WAF1/CIP1) promoter but not the basal promoter activity. In each transfection, 0.5 μg of reporter gene, 0.5 μg of AR, with or without 1 μg of BRCA1, were cotransfected into PC-3 cells. After 2 h of transfection, the medium was changed, and 10 nM DHT was added for another 30 h. ( B ) The endogenous p21 (WAF1/CIP1) expression was induced by DHT-AR and BRCA1 in MCF-7 cells. MCF-7 cells were seeded on 60-mm wells and cotransfected with 2 μg of AR with or without 4 μg of BRCA1 by SuperFect . After 2 h of transfection, the medium was changed, and 10 nM DHT was applied for another 30 h. In each experiment, 60 μg of whole-cell extract was applied for the Western blotting. ( C ) The p21 (WAF1/CIP1) protein is enhanced by DHT/AR and inhibited by hydroxyflutamide (HF) in PC-3(AR2) cells. The expression of protein p21 (WAF1/CIP1) can be induced by DHT, and this induction can be inhibited by 1 μM HF (lane 2 vs. lane 3). ( D ) The p21 (WAF1/CIP1) protein is enhanced by DHT/AR and BRCA1 in PC-3(AR2). PC-3(AR2) cells were cotransfected with or without 4 μg of BRCA1. After 2 h of transfection, the medium was changed, and 10 nM DHT was applied for another 30 h. In each experiment, 60 μg of whole-cell extract was applied for the Western blotting. ( E ) Cell growth is regulated by DHT/AR and BRCA1 in PC-3(AR2) cells. Duplicate PC-3(AR2) cells (as in D ) were applied to MTT assay for the relative cell number determination. ( F ) Dead cells were indicated as loss of cell membrane integrity assayed by PI inclusion. PC-3(AR2) cells were transfected with 4 μg of BRCA1. After 2 h of transfection, the medium was changed, and 10 nM DHT or vehicle was applied for another 4 days. The medium was changed on day 2. Attached cells were trypsinized and collected with floating cells, stained with 20 μg/ml PI on day 4. After 10 min of staining, the PI-positive cells were then counted under fluorescent microscope.

    Techniques Used: Activity Assay, Expressing, Transfection, Western Blot, MTT Assay, Staining, Microscopy

    5) Product Images from "Abnormal Mammary Gland Development and Growth Retardation in Female Mice and MCF7 Breast Cancer Cells Lacking Androgen Receptor"

    Article Title: Abnormal Mammary Gland Development and Growth Retardation in Female Mice and MCF7 Breast Cancer Cells Lacking Androgen Receptor

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20031233

    AR was essential for growth factor and estrogen signaling pathway. (A) Growth factor–induced cell proliferation was impaired in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. Cultures were incubated with RPMI 1640 media containing 0.2% serum treated with or without growth factors for 8 d. (B) The steady-state level of the active form of MAPK was lower in AR −/− MCF7 cells than that in AR +/+ MCF7 cells, when cells were cultured in the media containing 1% HI-FBS for 5 d (left). Growth factor–induced transcriptional activity of GAL4-Elk1 was diminished in AR −/− MCF7 cells (middle) and in AR +/+ MCF7 cells transfected with AR siRNA (right). (C) The reduced MAPK activity was restored by np-AR, which expresses full-length AR and is driven by natural AR promoter. np-AR synergistically enhanced EGF-induced GAL4-Elk1 transactivation. (D) The AR-FL–activated GAL4-Elk1 transactivation can be inhibited by a MAPK phosphatase (CL-100), a specific inhibitor U0126, dominant-negative Ras (Ras-DN), or Raf (Raf-DN). AR-FL , full-length WT AR. (E) Constitutively activated MEK1 (MEK-CA), Ras (Ras-CA), or Raf (Raf-CA), but not Rac (Rac-CA) or PI3K (p110 catalytic subunit), can block the suppressive effect of AR siRNA on the activity of GAL4-Elk1. (F) The transcriptional activity of ER is reduced in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. (G) The reduced ER activity in AR −/− MCF7 cells was restored by np-AR. pG5-Luc and ERE-Luc were the reporters for GAL4-Elk1 and ER, respectively. pRL-TK was used as an internal control. Transfections were performed using SuperFect according to the manufacturer's instructions. Values shown are the mean ± SD from at least four independent experiments.
    Figure Legend Snippet: AR was essential for growth factor and estrogen signaling pathway. (A) Growth factor–induced cell proliferation was impaired in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. Cultures were incubated with RPMI 1640 media containing 0.2% serum treated with or without growth factors for 8 d. (B) The steady-state level of the active form of MAPK was lower in AR −/− MCF7 cells than that in AR +/+ MCF7 cells, when cells were cultured in the media containing 1% HI-FBS for 5 d (left). Growth factor–induced transcriptional activity of GAL4-Elk1 was diminished in AR −/− MCF7 cells (middle) and in AR +/+ MCF7 cells transfected with AR siRNA (right). (C) The reduced MAPK activity was restored by np-AR, which expresses full-length AR and is driven by natural AR promoter. np-AR synergistically enhanced EGF-induced GAL4-Elk1 transactivation. (D) The AR-FL–activated GAL4-Elk1 transactivation can be inhibited by a MAPK phosphatase (CL-100), a specific inhibitor U0126, dominant-negative Ras (Ras-DN), or Raf (Raf-DN). AR-FL , full-length WT AR. (E) Constitutively activated MEK1 (MEK-CA), Ras (Ras-CA), or Raf (Raf-CA), but not Rac (Rac-CA) or PI3K (p110 catalytic subunit), can block the suppressive effect of AR siRNA on the activity of GAL4-Elk1. (F) The transcriptional activity of ER is reduced in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. (G) The reduced ER activity in AR −/− MCF7 cells was restored by np-AR. pG5-Luc and ERE-Luc were the reporters for GAL4-Elk1 and ER, respectively. pRL-TK was used as an internal control. Transfections were performed using SuperFect according to the manufacturer's instructions. Values shown are the mean ± SD from at least four independent experiments.

    Techniques Used: Incubation, Cell Culture, Activity Assay, Transfection, Dominant Negative Mutation, Blocking Assay

    6) Product Images from "Major Human Cytomegalovirus Structural Protein pp65 (ppUL83) Prevents Interferon Response Factor 3 Activation in the Interferon Response"

    Article Title: Major Human Cytomegalovirus Structural Protein pp65 (ppUL83) Prevents Interferon Response Factor 3 Activation in the Interferon Response

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.20.10995-11006.2004

    Immunofluorescence analysis of IRF-3 localization in pp65-transduced cells. Immunofluorescence assays show IRF-3 localization 4 h after exposure to induction with pcDNA3-EYFP-loaded Superfect in control HFs (A) and pp65-transduced HFs (B) or at 4 hpi with pp65 mutant (mut) virus in pp65-transduced HFs (C). Overlays of IRF-3 stain with Hoechst 44432 show nuclei (D to F). Immunofluorescence assay shows pp65 localization in pp65-transduced HFs (G).
    Figure Legend Snippet: Immunofluorescence analysis of IRF-3 localization in pp65-transduced cells. Immunofluorescence assays show IRF-3 localization 4 h after exposure to induction with pcDNA3-EYFP-loaded Superfect in control HFs (A) and pp65-transduced HFs (B) or at 4 hpi with pp65 mutant (mut) virus in pp65-transduced HFs (C). Overlays of IRF-3 stain with Hoechst 44432 show nuclei (D to F). Immunofluorescence assay shows pp65 localization in pp65-transduced HFs (G).

    Techniques Used: Immunofluorescence, Mutagenesis, Staining

    7) Product Images from "Triazine dendrimers as non-viral gene delivery systems: Effects of molecular structure on biological activity"

    Article Title: Triazine dendrimers as non-viral gene delivery systems: Effects of molecular structure on biological activity

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc900243r

    Transfection efficiency of the dendrimers at various N/P ratios in (A) L929 and (B) MeWo cells are given as relative light units detected by luciferase assay. F2-1 and Superfect exhibited significantly (***p
    Figure Legend Snippet: Transfection efficiency of the dendrimers at various N/P ratios in (A) L929 and (B) MeWo cells are given as relative light units detected by luciferase assay. F2-1 and Superfect exhibited significantly (***p

    Techniques Used: Transfection, Luciferase

    8) Product Images from "Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir"

    Article Title: Transcription Factor SP2 Enhanced the Expression of Cd14 in Colitis-Susceptible C3H/HeJBir

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155821

    Transfection system establishment. A Western blot analysis of CD14 expression in untreated and LPS stimulated murine RAW264.7 macrophages and NIH/3T3 fibroblasts, β-actin as endogeous control. Transfection efficiency test by determination of GFP-positive RAW264.7 cells by cyto fluorescence 24 hours after transfection with B Xtreme Gene HP, C Xtreme Gene 9 DNA, D Superfect, E Polyfect, and F by β-galactosidase assay, ***: p
    Figure Legend Snippet: Transfection system establishment. A Western blot analysis of CD14 expression in untreated and LPS stimulated murine RAW264.7 macrophages and NIH/3T3 fibroblasts, β-actin as endogeous control. Transfection efficiency test by determination of GFP-positive RAW264.7 cells by cyto fluorescence 24 hours after transfection with B Xtreme Gene HP, C Xtreme Gene 9 DNA, D Superfect, E Polyfect, and F by β-galactosidase assay, ***: p

    Techniques Used: Transfection, Western Blot, Expressing, Fluorescence

    9) Product Images from "Homeoproteins CDP and SATB1 Interact: Potential for Tissue-Specific Regulation"

    Article Title: Homeoproteins CDP and SATB1 Interact: Potential for Tissue-Specific Regulation

    Journal: Molecular and Cellular Biology

    doi:

    Overexpression of CDP in Jurkat cells elevates expression from the MMTV LTR. (A) Overexpression of CDP in Jurkat cells by electroporation. Transfections of the CDP expression vector (15 μg) were performed in triplicate, and results are expressed as an average increase over the value determined from the average of three determinations of the CDP-negative control vector (assigned a value of 1). Error bar indicates the standard deviation from the mean. (B) Overexpression of CDP in Jurkat cells by using the SuperFect transfection method. Values were calculated as described for panel A. Addition of more DNA (from any source) in the transfection assays resulted in lower reporter gene activity.
    Figure Legend Snippet: Overexpression of CDP in Jurkat cells elevates expression from the MMTV LTR. (A) Overexpression of CDP in Jurkat cells by electroporation. Transfections of the CDP expression vector (15 μg) were performed in triplicate, and results are expressed as an average increase over the value determined from the average of three determinations of the CDP-negative control vector (assigned a value of 1). Error bar indicates the standard deviation from the mean. (B) Overexpression of CDP in Jurkat cells by using the SuperFect transfection method. Values were calculated as described for panel A. Addition of more DNA (from any source) in the transfection assays resulted in lower reporter gene activity.

    Techniques Used: Over Expression, Expressing, Electroporation, Transfection, Plasmid Preparation, Negative Control, Standard Deviation, Activity Assay

    10) Product Images from "Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway"

    Article Title: Efficient siRNA delivery and gene silencing using a lipopolypeptide hybrid vector mediated by a caveolae-mediated and temperature-dependent endocytic pathway

    Journal: Journal of Nanobiotechnology

    doi: 10.1186/s12951-019-0444-8

    Comparative transfection efficiency of FuGENE HD (FuGENE), X-tremeGENE (X-treme), SuperFect (Super), Lipofectamine 2000 (L-2000), Lipofectamine RNAiMAX (L-iMAX), INT, and mTat/PEI/INT using siRNA targeting β - actin in HSC-3 cells. β - actin mRNA was measured by QRT-PCR and then % remaining β - actin mRNA expression was calculated based on control as 100% ( a ). Cell viability of each of the reagent groups in HaCaT cells were evaluated by MTT assay under the same siRNA and transfection reagent volume ( b ). * p
    Figure Legend Snippet: Comparative transfection efficiency of FuGENE HD (FuGENE), X-tremeGENE (X-treme), SuperFect (Super), Lipofectamine 2000 (L-2000), Lipofectamine RNAiMAX (L-iMAX), INT, and mTat/PEI/INT using siRNA targeting β - actin in HSC-3 cells. β - actin mRNA was measured by QRT-PCR and then % remaining β - actin mRNA expression was calculated based on control as 100% ( a ). Cell viability of each of the reagent groups in HaCaT cells were evaluated by MTT assay under the same siRNA and transfection reagent volume ( b ). * p

    Techniques Used: Transfection, Quantitative RT-PCR, Expressing, MTT Assay

    11) Product Images from "GRK2 is an endogenous protein inhibitor of the insulin signaling pathway for glucose transport stimulation"

    Article Title: GRK2 is an endogenous protein inhibitor of the insulin signaling pathway for glucose transport stimulation

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600297

    Effects of the RGS domain of GRK2 on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. ( A ) Plasmid expression vectors of WT-, KD-, or deletion mutant (Delta) GRK2 were transfected in HIRc-B cells using SuperFECT as described in Materials and methods. At 48 h after transfection, these cells were lysed and total cell lysates were analyzed by Western blotting using anti-GRK2 antibody as described in Materials and methods. These experiments were performed three times, and a representative result is shown. ( B ) Plasmid expression vectors of WT-GRK2, KD-GRK2, delta-GRK2, or control ERK1 (HA-ERK1) were microinjected into the nuclei of 3T3-L1 adipocytes on coverslips. At 24 h after microinjection, 3T3-L1 adipocytes were serum starved for 4 h and were stimulated with 1.7 nM insulin for 20 min. GLUT4 was stained as described in Materials and methods. The cells expressing exogenous GRKs or ERK1 were detected by staining with anti-6X-His antibody or anti-HA antibody, respectively. The percentage of cells positive for GLUT4 translocation was calculated by counting at least 100 cells at each point. The data are the mean±s.e. from three independent experiments.
    Figure Legend Snippet: Effects of the RGS domain of GRK2 on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. ( A ) Plasmid expression vectors of WT-, KD-, or deletion mutant (Delta) GRK2 were transfected in HIRc-B cells using SuperFECT as described in Materials and methods. At 48 h after transfection, these cells were lysed and total cell lysates were analyzed by Western blotting using anti-GRK2 antibody as described in Materials and methods. These experiments were performed three times, and a representative result is shown. ( B ) Plasmid expression vectors of WT-GRK2, KD-GRK2, delta-GRK2, or control ERK1 (HA-ERK1) were microinjected into the nuclei of 3T3-L1 adipocytes on coverslips. At 24 h after microinjection, 3T3-L1 adipocytes were serum starved for 4 h and were stimulated with 1.7 nM insulin for 20 min. GLUT4 was stained as described in Materials and methods. The cells expressing exogenous GRKs or ERK1 were detected by staining with anti-6X-His antibody or anti-HA antibody, respectively. The percentage of cells positive for GLUT4 translocation was calculated by counting at least 100 cells at each point. The data are the mean±s.e. from three independent experiments.

    Techniques Used: Translocation Assay, Plasmid Preparation, Expressing, Mutagenesis, Transfection, Western Blot, Staining

    12) Product Images from "Convergence of two repressors through heterodimer formation of androgen receptor and testicular orphan receptor-4: A unique signaling pathway in the steroid receptor superfamily"

    Article Title: Convergence of two repressors through heterodimer formation of androgen receptor and testicular orphan receptor-4: A unique signaling pathway in the steroid receptor superfamily

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

    doi:

    AR repression of TR4-target gene expression. ( A ) AR repression of TR4-mediated DR4-TK-CAT and DR1-CNTFR-I5-LUC transcriptional activity. We cotransfected 500 ng of reporter plasmids (DR4-TK-CAT and DR1-CNTFR-I5-LUC) with 200 ng of pCMX-TR4 and increasing amounts of pCMV-AR (200, 600, and 1,200 ng), pSG5GR (1,200 ng), or pSG5 PR (1,200 ng) by using the SuperFect transfection kit (Qiagen). ( B ) AR repression of TR4-mediated HBV gene expression. The reporter plasmid CpFL(4)-LUC, which contains the HBV core promoter (Cp) sequence located between −34 and −7 (nucleotide coordinates 1751 and 1778 derived from the GenBank database) was shown above. The arrows indicate the DR1 motif in HBV core promoter. HepG2 cells were cotransfected with 1.5 μg of CpFL(4)-LUC reporter and 0.5 μg of pCMX-TR4, with increasing amounts of pCMV-AR (0.5, 2.5, and 5 μg) by modified calcium phosphate precipitation method. The relative reporter gene activities were compared with the CAT activities (or luciferase activities) with vector alone. To normalize the transfection efficiency, the β-galactosidase expression vector and pRL-TK were cotransfected in the CAT assay and in the dual-luciferase reporter assay system (Promega), respectively.
    Figure Legend Snippet: AR repression of TR4-target gene expression. ( A ) AR repression of TR4-mediated DR4-TK-CAT and DR1-CNTFR-I5-LUC transcriptional activity. We cotransfected 500 ng of reporter plasmids (DR4-TK-CAT and DR1-CNTFR-I5-LUC) with 200 ng of pCMX-TR4 and increasing amounts of pCMV-AR (200, 600, and 1,200 ng), pSG5GR (1,200 ng), or pSG5 PR (1,200 ng) by using the SuperFect transfection kit (Qiagen). ( B ) AR repression of TR4-mediated HBV gene expression. The reporter plasmid CpFL(4)-LUC, which contains the HBV core promoter (Cp) sequence located between −34 and −7 (nucleotide coordinates 1751 and 1778 derived from the GenBank database) was shown above. The arrows indicate the DR1 motif in HBV core promoter. HepG2 cells were cotransfected with 1.5 μg of CpFL(4)-LUC reporter and 0.5 μg of pCMX-TR4, with increasing amounts of pCMV-AR (0.5, 2.5, and 5 μg) by modified calcium phosphate precipitation method. The relative reporter gene activities were compared with the CAT activities (or luciferase activities) with vector alone. To normalize the transfection efficiency, the β-galactosidase expression vector and pRL-TK were cotransfected in the CAT assay and in the dual-luciferase reporter assay system (Promega), respectively.

    Techniques Used: Expressing, Activity Assay, Transfection, Plasmid Preparation, Sequencing, Derivative Assay, Modification, Luciferase, Reporter Assay

    TR4 repression of AR-mediated transcriptional activity. ( A ) Five hundred nanograms of MMTV-Luc was cotransfected with 40 ng of pCMV-AR (lanes 2 and 3) with increasing amounts of pCMX-TR4 (400, 800, and 1,200 ng). ( B ) Five hundred nanograms of PSA-Luc was cotransfected with 40 ng pCMV-AR (lanes 2 and 3) with increasing amounts of pCMX-TR4 (400 and 1,200 ng). After 24 hr transfection, cells were treated with 10 nM of DHT. After 16–18 hr incubation, cells were harvested for dual-luciferase reporter assay. ( C ) Northern blotting analysis of PSA transcripts in LNCaP cells. Total RNA (25 μg) from LNCaP cells, which were transfected with either pCMX-TR4 or pCMX vector by using SuperFect (Qiagen), was applied into a formamide RNA gel, transferred onto a nylon membrane, and hybridized with a 32 P-PSA gene fragment from the exon 3. β-actin was used as an internal control.
    Figure Legend Snippet: TR4 repression of AR-mediated transcriptional activity. ( A ) Five hundred nanograms of MMTV-Luc was cotransfected with 40 ng of pCMV-AR (lanes 2 and 3) with increasing amounts of pCMX-TR4 (400, 800, and 1,200 ng). ( B ) Five hundred nanograms of PSA-Luc was cotransfected with 40 ng pCMV-AR (lanes 2 and 3) with increasing amounts of pCMX-TR4 (400 and 1,200 ng). After 24 hr transfection, cells were treated with 10 nM of DHT. After 16–18 hr incubation, cells were harvested for dual-luciferase reporter assay. ( C ) Northern blotting analysis of PSA transcripts in LNCaP cells. Total RNA (25 μg) from LNCaP cells, which were transfected with either pCMX-TR4 or pCMX vector by using SuperFect (Qiagen), was applied into a formamide RNA gel, transferred onto a nylon membrane, and hybridized with a 32 P-PSA gene fragment from the exon 3. β-actin was used as an internal control.

    Techniques Used: Activity Assay, Transfection, Incubation, Luciferase, Reporter Assay, Northern Blot, Plasmid Preparation

    13) Product Images from "A combined approach of hollow microneedles and nanocarriers for skin immunization with plasmid DNA encoding ovalbumin"

    Article Title: A combined approach of hollow microneedles and nanocarriers for skin immunization with plasmid DNA encoding ovalbumin

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S125945

    Gel retardation of ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect:pOVA complexes to confirm the positive charges of the complexes at different weight ratios. Abbreviations: PEI, polyethylenimine; pOVA, plasmid DNA encoding ovalbumin.
    Figure Legend Snippet: Gel retardation of ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect:pOVA complexes to confirm the positive charges of the complexes at different weight ratios. Abbreviations: PEI, polyethylenimine; pOVA, plasmid DNA encoding ovalbumin.

    Techniques Used: Electrophoretic Mobility Shift Assay, Plasmid Preparation

    Cell viability of HeLa cells after transfected with ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect:pOVA complexes at pH 7.4. Notes: Each value was presented as mean ± SD of triplicate samples. * P
    Figure Legend Snippet: Cell viability of HeLa cells after transfected with ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect:pOVA complexes at pH 7.4. Notes: Each value was presented as mean ± SD of triplicate samples. * P

    Techniques Used: Transfection

    In vitro transfection efficiency in HeLa cells at varying weight ratios of ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect: pOVA complexes. Note: * P
    Figure Legend Snippet: In vitro transfection efficiency in HeLa cells at varying weight ratios of ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect: pOVA complexes. Note: * P

    Techniques Used: In Vitro, Transfection

    The particle size and zeta potential for varying weight ratios of ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect:pOVA complexes. Abbreviations: PEI, polyethylenimine; pOVA, plasmid DNA encoding ovalbumin.
    Figure Legend Snippet: The particle size and zeta potential for varying weight ratios of ( A ) PEI:pOVA complexes, ( B ) Lipofectamine 2000:pOVA complexes, and ( C ) Superfect:pOVA complexes. Abbreviations: PEI, polyethylenimine; pOVA, plasmid DNA encoding ovalbumin.

    Techniques Used: Plasmid Preparation

    In vitro permeation profiles of pOVA across the skin with ( A ) various delivery devices: (●) untreated skin, (○) EP patch, (▼) solid MN patch, (△) combined solid MN and EP patch, (■) hollow MN, ( B ) hollow MN: (■) naked pOVA, different cationic nanocarriers, including (◊) PEI:pOVA complexes, (▲) Lipofectamine 2000:pOVA complexes and (□) Superfect:pOVA complexes, ( C ) the cumulative amount at 24 h of all groups. Notes: * P
    Figure Legend Snippet: In vitro permeation profiles of pOVA across the skin with ( A ) various delivery devices: (●) untreated skin, (○) EP patch, (▼) solid MN patch, (△) combined solid MN and EP patch, (■) hollow MN, ( B ) hollow MN: (■) naked pOVA, different cationic nanocarriers, including (◊) PEI:pOVA complexes, (▲) Lipofectamine 2000:pOVA complexes and (□) Superfect:pOVA complexes, ( C ) the cumulative amount at 24 h of all groups. Notes: * P

    Techniques Used: In Vitro

    Gel retardation assay. Notes: Lane 1, DNA marker; lane 2, naked pOVA without serum; lane 3, naked pOVA in the presence of 10% serum of BALB/c mice for 6 h; lane 4, PEI:pOVA complexes (1:1) without serum; lane 5, PEI:pOVA complexes (1:1) in the presence of 10% serum; lane 6, Lipofectamine 2000:pOVA complexes (2:1) without serum; lane 7, Lipofectamine 2000:pOVA complexes (2:1) in the presence of 10% serum; lane 8, Superfect:pOVA complexes (6:1) without serum; and lane 9, Superfect: pOVA complexes (6:1) in the presence of 10% serum. Abbreviations: pOVA, plasmid DNA encoding ovalbumin; PEI, polyethylenimine.
    Figure Legend Snippet: Gel retardation assay. Notes: Lane 1, DNA marker; lane 2, naked pOVA without serum; lane 3, naked pOVA in the presence of 10% serum of BALB/c mice for 6 h; lane 4, PEI:pOVA complexes (1:1) without serum; lane 5, PEI:pOVA complexes (1:1) in the presence of 10% serum; lane 6, Lipofectamine 2000:pOVA complexes (2:1) without serum; lane 7, Lipofectamine 2000:pOVA complexes (2:1) in the presence of 10% serum; lane 8, Superfect:pOVA complexes (6:1) without serum; and lane 9, Superfect: pOVA complexes (6:1) in the presence of 10% serum. Abbreviations: pOVA, plasmid DNA encoding ovalbumin; PEI, polyethylenimine.

    Techniques Used: Electrophoretic Mobility Shift Assay, Marker, Mouse Assay, Plasmid Preparation

    14) Product Images from "Increased Acetylation in the DNA-binding Domain of TR4 Nuclear Receptor by the Coregulator ARA55 Leads to Suppression of TR4 Transactivation *"

    Article Title: Increased Acetylation in the DNA-binding Domain of TR4 Nuclear Receptor by the Coregulator ARA55 Leads to Suppression of TR4 Transactivation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.208181

    ARA55 enhances AR transcriptional activity but inhibits TR4 activity. A , ARA55 enhances AR transcriptional activity but inhibits TR4 transcriptional activity. Left panel , 293T cells were cotransfected with pSG5-AR, MMTV-luc, and pCDNA3-flagARA55 as indicated (the ratio of AR to ARA55 was 1:3) using Superfect according to the manual instructions. After 16 h, the cells were treated with vehicle or dihydrotestosterone for an additional day and then harvested for AR luciferase assay. Right panel , 293T cells were cotransfected with pCMX-TR4, HCR-luc, and pcDNA3-flagARA55 as indicated (the ratio of TR4 to ARA55 was 1:3). After 24 h, the cells were harvested, and TR4 luciferase activity was examined. B , ARA55 reduces the mRNA expression level of CD36, a target gene of TR4. Hep1-6 cells were cotransfected with pCDNA3-flagTR4 and pCDNA4-HisARA55 as indicated. After 24 h, the cells were harvested, and CD36 mRNA levels were examined by real time RT-PCR. 18 S rRNA level was used as the internal control, and the CD36 mRNA expression in control cells (with empty vector) was set as 1. The data represent the means ± S.D. of triplicate samples. C , ARA55 reduces the protein level of CD36. Hep1-6 cells were cotransfected with pCDNA3flagTR4 and pCDNA4HisARA55 as indicated. After 24 h, the cells were harvested, and the protein levels of CD36 as well as TR4 and ARA55 were examined by Western blot analysis. GAPDH served as the loading control. DHT , dihydrotestosterone; IB , immunoblot.
    Figure Legend Snippet: ARA55 enhances AR transcriptional activity but inhibits TR4 activity. A , ARA55 enhances AR transcriptional activity but inhibits TR4 transcriptional activity. Left panel , 293T cells were cotransfected with pSG5-AR, MMTV-luc, and pCDNA3-flagARA55 as indicated (the ratio of AR to ARA55 was 1:3) using Superfect according to the manual instructions. After 16 h, the cells were treated with vehicle or dihydrotestosterone for an additional day and then harvested for AR luciferase assay. Right panel , 293T cells were cotransfected with pCMX-TR4, HCR-luc, and pcDNA3-flagARA55 as indicated (the ratio of TR4 to ARA55 was 1:3). After 24 h, the cells were harvested, and TR4 luciferase activity was examined. B , ARA55 reduces the mRNA expression level of CD36, a target gene of TR4. Hep1-6 cells were cotransfected with pCDNA3-flagTR4 and pCDNA4-HisARA55 as indicated. After 24 h, the cells were harvested, and CD36 mRNA levels were examined by real time RT-PCR. 18 S rRNA level was used as the internal control, and the CD36 mRNA expression in control cells (with empty vector) was set as 1. The data represent the means ± S.D. of triplicate samples. C , ARA55 reduces the protein level of CD36. Hep1-6 cells were cotransfected with pCDNA3flagTR4 and pCDNA4HisARA55 as indicated. After 24 h, the cells were harvested, and the protein levels of CD36 as well as TR4 and ARA55 were examined by Western blot analysis. GAPDH served as the loading control. DHT , dihydrotestosterone; IB , immunoblot.

    Techniques Used: Activity Assay, Luciferase, Expressing, Quantitative RT-PCR, Plasmid Preparation, Western Blot

    15) Product Images from "Triazine dendrimers as non-viral gene delivery systems: Effects of molecular structure on biological activity"

    Article Title: Triazine dendrimers as non-viral gene delivery systems: Effects of molecular structure on biological activity

    Journal: Bioconjugate chemistry

    doi: 10.1021/bc900243r

    Transfection efficiency of the dendrimers at various N/P ratios in (A) L929 and (B) MeWo cells are given as relative light units detected by luciferase assay. F2-1 and Superfect exhibited significantly (***p
    Figure Legend Snippet: Transfection efficiency of the dendrimers at various N/P ratios in (A) L929 and (B) MeWo cells are given as relative light units detected by luciferase assay. F2-1 and Superfect exhibited significantly (***p

    Techniques Used: Transfection, Luciferase

    16) Product Images from "Structure and Specificity of GATA Proteins in Th2 Development"

    Article Title: Structure and Specificity of GATA Proteins in Th2 Development

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.8.2716-2725.2001

    ). Cells were sorted 7 days after the first activation, restimulated with ovalbumin (0.5 mg/ml) and irradiated BALB/c splenocytes (2.5 × 10 6 ). Data are presented as in panel B. (D) Expression of murine stem cell virus (MSCV) long terminal repeat (LTR)-driven GATA-3, c-Maf, and JunB. 293 cells were transiently transfected with 20 μg of GATA3-RV, cmaf-RV, and JunB-RV using Superfect (Quiagen). After 48 h, cells lysates were electrophoresed by SDS–12% PAGE, transferred to nitrocellulose, and analyzed by Western analysis. GATA-3 was detected using antibody HG3-31 (Santa Cruz), c-Maf was detected using M173 (Santa Cruz), JunB was detected using N17 (Santa Cruz), and Stat1 was detected using E23 (Santa Cruz).
    Figure Legend Snippet: ). Cells were sorted 7 days after the first activation, restimulated with ovalbumin (0.5 mg/ml) and irradiated BALB/c splenocytes (2.5 × 10 6 ). Data are presented as in panel B. (D) Expression of murine stem cell virus (MSCV) long terminal repeat (LTR)-driven GATA-3, c-Maf, and JunB. 293 cells were transiently transfected with 20 μg of GATA3-RV, cmaf-RV, and JunB-RV using Superfect (Quiagen). After 48 h, cells lysates were electrophoresed by SDS–12% PAGE, transferred to nitrocellulose, and analyzed by Western analysis. GATA-3 was detected using antibody HG3-31 (Santa Cruz), c-Maf was detected using M173 (Santa Cruz), JunB was detected using N17 (Santa Cruz), and Stat1 was detected using E23 (Santa Cruz).

    Techniques Used: Activation Assay, Irradiation, Expressing, Transfection, Polyacrylamide Gel Electrophoresis, Western Blot

    17) Product Images from "Isolation and characterization of the activated B-cell factor 1 homolog in Caenorhabditis elegans"

    Article Title: Isolation and characterization of the activated B-cell factor 1 homolog in Caenorhabditis elegans

    Journal: Nucleic Acids Research

    doi:

    SK residues found within the first helix of the bHLH domain of CeABF-1 help mediate repression of E47 activity. HeLa S3 cells were transiently transfected with 1 µg of (µE5-µE3-µE2) 4 -Luc reporter vector and different combinations of the E47 expression vector (0.5 µg), FLAG-CeABF-1, and FLAG-CeABF-1 SK-NE expression plasmids (0.25 µg). Twenty-five microliters of Superfect reagent was used for each transfection and equal amounts of protein extract were assayed for firefly luciferase activity. Experiments were performed in triplicate and represent the means of two independent assays. Luciferase activity was normalized to β-galactosidase levels as a control for transfection efficiency. The mean of the firefly luciferase value for E47 + CeABF-1 was 60 248 light units and E47 + CeABF-1 SK-NE was 198 818 light units.
    Figure Legend Snippet: SK residues found within the first helix of the bHLH domain of CeABF-1 help mediate repression of E47 activity. HeLa S3 cells were transiently transfected with 1 µg of (µE5-µE3-µE2) 4 -Luc reporter vector and different combinations of the E47 expression vector (0.5 µg), FLAG-CeABF-1, and FLAG-CeABF-1 SK-NE expression plasmids (0.25 µg). Twenty-five microliters of Superfect reagent was used for each transfection and equal amounts of protein extract were assayed for firefly luciferase activity. Experiments were performed in triplicate and represent the means of two independent assays. Luciferase activity was normalized to β-galactosidase levels as a control for transfection efficiency. The mean of the firefly luciferase value for E47 + CeABF-1 was 60 248 light units and E47 + CeABF-1 SK-NE was 198 818 light units.

    Techniques Used: Activity Assay, Transfection, Plasmid Preparation, Expressing, Luciferase

    CeABF-1 functions as a transcriptional repressor in mammalian cells similar to human ABF-1. HeLa S3 cells were transiently transfected with 1 µg of (µE5-µE3-µE2) 4 -Luc reporter plasmid and different combinations of either the E47 expression vector (0.5 µg) with expression vector FLAG-human ABF-1, FLAG-CeABF-1, CeABF-1 N-73 or CeABF-1 C-97 (0.25 µg). Twenty-five microliters of Superfect reagent was used for each transfection. Equal amounts of protein extract were assayed for firefly luciferase activity. Experiments were performed in triplicates and run at least three independent times. Luciferase activity was normalized to β-galactosidase levels as a control for transfection efficiency.
    Figure Legend Snippet: CeABF-1 functions as a transcriptional repressor in mammalian cells similar to human ABF-1. HeLa S3 cells were transiently transfected with 1 µg of (µE5-µE3-µE2) 4 -Luc reporter plasmid and different combinations of either the E47 expression vector (0.5 µg) with expression vector FLAG-human ABF-1, FLAG-CeABF-1, CeABF-1 N-73 or CeABF-1 C-97 (0.25 µg). Twenty-five microliters of Superfect reagent was used for each transfection. Equal amounts of protein extract were assayed for firefly luciferase activity. Experiments were performed in triplicates and run at least three independent times. Luciferase activity was normalized to β-galactosidase levels as a control for transfection efficiency.

    Techniques Used: Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay

    18) Product Images from "Abnormal Mammary Gland Development and Growth Retardation in Female Mice and MCF7 Breast Cancer Cells Lacking Androgen Receptor"

    Article Title: Abnormal Mammary Gland Development and Growth Retardation in Female Mice and MCF7 Breast Cancer Cells Lacking Androgen Receptor

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20031233

    AR was essential for growth factor and estrogen signaling pathway. (A) Growth factor–induced cell proliferation was impaired in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. Cultures were incubated with RPMI 1640 media containing 0.2% serum treated with or without growth factors for 8 d. (B) The steady-state level of the active form of MAPK was lower in AR −/− MCF7 cells than that in AR +/+ MCF7 cells, when cells were cultured in the media containing 1% HI-FBS for 5 d (left). Growth factor–induced transcriptional activity of GAL4-Elk1 was diminished in AR −/− MCF7 cells (middle) and in AR +/+ MCF7 cells transfected with AR siRNA (right). (C) The reduced MAPK activity was restored by np-AR, which expresses full-length AR and is driven by natural AR promoter. np-AR synergistically enhanced EGF-induced GAL4-Elk1 transactivation. (D) The AR-FL–activated GAL4-Elk1 transactivation can be inhibited by a MAPK phosphatase (CL-100), a specific inhibitor U0126, dominant-negative Ras (Ras-DN), or Raf (Raf-DN). AR-FL , full-length WT AR. (E) Constitutively activated MEK1 (MEK-CA), Ras (Ras-CA), or Raf (Raf-CA), but not Rac (Rac-CA) or PI3K (p110 catalytic subunit), can block the suppressive effect of AR siRNA on the activity of GAL4-Elk1. (F) The transcriptional activity of ER is reduced in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. (G) The reduced ER activity in AR −/− MCF7 cells was restored by np-AR. pG5-Luc and ERE-Luc were the reporters for GAL4-Elk1 and ER, respectively. pRL-TK was used as an internal control. Transfections were performed using SuperFect according to the manufacturer's instructions. Values shown are the mean ± SD from at least four independent experiments.
    Figure Legend Snippet: AR was essential for growth factor and estrogen signaling pathway. (A) Growth factor–induced cell proliferation was impaired in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. Cultures were incubated with RPMI 1640 media containing 0.2% serum treated with or without growth factors for 8 d. (B) The steady-state level of the active form of MAPK was lower in AR −/− MCF7 cells than that in AR +/+ MCF7 cells, when cells were cultured in the media containing 1% HI-FBS for 5 d (left). Growth factor–induced transcriptional activity of GAL4-Elk1 was diminished in AR −/− MCF7 cells (middle) and in AR +/+ MCF7 cells transfected with AR siRNA (right). (C) The reduced MAPK activity was restored by np-AR, which expresses full-length AR and is driven by natural AR promoter. np-AR synergistically enhanced EGF-induced GAL4-Elk1 transactivation. (D) The AR-FL–activated GAL4-Elk1 transactivation can be inhibited by a MAPK phosphatase (CL-100), a specific inhibitor U0126, dominant-negative Ras (Ras-DN), or Raf (Raf-DN). AR-FL , full-length WT AR. (E) Constitutively activated MEK1 (MEK-CA), Ras (Ras-CA), or Raf (Raf-CA), but not Rac (Rac-CA) or PI3K (p110 catalytic subunit), can block the suppressive effect of AR siRNA on the activity of GAL4-Elk1. (F) The transcriptional activity of ER is reduced in AR −/− MCF7 cells, compared with AR +/+ MCF7 cells. (G) The reduced ER activity in AR −/− MCF7 cells was restored by np-AR. pG5-Luc and ERE-Luc were the reporters for GAL4-Elk1 and ER, respectively. pRL-TK was used as an internal control. Transfections were performed using SuperFect according to the manufacturer's instructions. Values shown are the mean ± SD from at least four independent experiments.

    Techniques Used: Incubation, Cell Culture, Activity Assay, Transfection, Dominant Negative Mutation, Blocking Assay

    19) Product Images from "Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications"

    Article Title: Environmental parameters influence non-viral transfection of human mesenchymal stem cells for tissue engineering applications

    Journal: Cell and Tissue Research

    doi: 10.1007/s00441-011-1297-0

    Effect of transfection reagents on transfection efficiency and hMSC viability. a) Transfection efficiency of passage 6 hMSCs with pDNA complexed with JetPEI, Superfect, or 60k branched PEI 1 and 2 days post transfection normalized to PEI’s transfection
    Figure Legend Snippet: Effect of transfection reagents on transfection efficiency and hMSC viability. a) Transfection efficiency of passage 6 hMSCs with pDNA complexed with JetPEI, Superfect, or 60k branched PEI 1 and 2 days post transfection normalized to PEI’s transfection

    Techniques Used: Transfection

    20) Product Images from "GRK2 is an endogenous protein inhibitor of the insulin signaling pathway for glucose transport stimulation"

    Article Title: GRK2 is an endogenous protein inhibitor of the insulin signaling pathway for glucose transport stimulation

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7600297

    Effects of the RGS domain of GRK2 on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. ( A ) Plasmid expression vectors of WT-, KD-, or deletion mutant (Delta) GRK2 were transfected in HIRc-B cells using SuperFECT as described in Materials and methods. At 48 h after transfection, these cells were lysed and total cell lysates were analyzed by Western blotting using anti-GRK2 antibody as described in Materials and methods. These experiments were performed three times, and a representative result is shown. ( B ) Plasmid expression vectors of WT-GRK2, KD-GRK2, delta-GRK2, or control ERK1 (HA-ERK1) were microinjected into the nuclei of 3T3-L1 adipocytes on coverslips. At 24 h after microinjection, 3T3-L1 adipocytes were serum starved for 4 h and were stimulated with 1.7 nM insulin for 20 min. GLUT4 was stained as described in Materials and methods. The cells expressing exogenous GRKs or ERK1 were detected by staining with anti-6X-His antibody or anti-HA antibody, respectively. The percentage of cells positive for GLUT4 translocation was calculated by counting at least 100 cells at each point. The data are the mean±s.e. from three independent experiments.
    Figure Legend Snippet: Effects of the RGS domain of GRK2 on insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. ( A ) Plasmid expression vectors of WT-, KD-, or deletion mutant (Delta) GRK2 were transfected in HIRc-B cells using SuperFECT as described in Materials and methods. At 48 h after transfection, these cells were lysed and total cell lysates were analyzed by Western blotting using anti-GRK2 antibody as described in Materials and methods. These experiments were performed three times, and a representative result is shown. ( B ) Plasmid expression vectors of WT-GRK2, KD-GRK2, delta-GRK2, or control ERK1 (HA-ERK1) were microinjected into the nuclei of 3T3-L1 adipocytes on coverslips. At 24 h after microinjection, 3T3-L1 adipocytes were serum starved for 4 h and were stimulated with 1.7 nM insulin for 20 min. GLUT4 was stained as described in Materials and methods. The cells expressing exogenous GRKs or ERK1 were detected by staining with anti-6X-His antibody or anti-HA antibody, respectively. The percentage of cells positive for GLUT4 translocation was calculated by counting at least 100 cells at each point. The data are the mean±s.e. from three independent experiments.

    Techniques Used: Translocation Assay, Plasmid Preparation, Expressing, Mutagenesis, Transfection, Western Blot, Staining

    21) Product Images from "Human T-Cell Lymphotropic/Leukemia Virus Type 1 Tax Abrogates p53-Induced Cell Cycle Arrest and Apoptosis through Its CREB/ATF Functional Domain"

    Article Title: Human T-Cell Lymphotropic/Leukemia Virus Type 1 Tax Abrogates p53-Induced Cell Cycle Arrest and Apoptosis through Its CREB/ATF Functional Domain

    Journal: Journal of Virology

    doi:

    p53 is not transcriptionally functional in HTLV-1-infected cell lines. U2OS cells were transfected by the Ca 2 PO 4 precipitation method with 1 μg of reporter plasmid and 1 μg of TK-renilla-Luc, an internal control plasmid to normalize transfection. Lysates were assayed for firefly luciferase and Renilla luciferase by using a dual assay kit (Promega). HTLV-1-infected cell lines were transfected by the DEAE-Dextran method with 1 μg each of the above-mentioned plasmids. Jurkat cells were transfected with Superfect with the same amounts of DNA as for the other cell lines. Error bars show the standard deviations of four independent experiments and are normalized for protein amount. (A) Fold induction by endogenous p53 activity on the p53-responsive plasmid PG13-Luc. The control plasmid was a construct containing a basal promoter expressing the firefly luciferase gene that contained no inducible element. U2OS expresses wild-type p53 and shows strong activation of PG13-Luc. Jurkat expresses a nonfunctional p53 protein and has no activity on this construct. All four of the HTLV-1-infected cell lines show no activity on PG13-Luc, even though they express high levels of wild-type p53. Both IL-2-dependent and -independent lines are negative for p53 transactivation. (B) Fold induction on the HTLV-1-LTR-Luc construct. This plasmid contains the entire HTLV-1 long terminal repeat cloned upstream of a basal promoter present in the PGL3-Luc construct and is strongly activated by HTLV-1 Tax. All four HTLV-1-infected cell lines activate this reporter, but neither U2OS nor Jurkat transactivate this construct to any extent. (C) Relative luciferase activity on the TK-renilla-Luc construct in the different cell lines. (D) Western blot analysis for the presence of endogenous p53 and Tax. Forty micrograms of protein lysate from luciferase assays was run on SDS–10% PAGE gels, and proteins were detected by chemiluminescence. The HTLV-1-infected cells express at least as much p53 protein as U2OS but show no activity on the PG13-Luc reporter gene (panel A). (E) Cells were mounted on slides by using cytospin funnels and were fixed for 10 min with 2% paraformaldehyde. Indirect immunofluorescence analysis was performed with the anti-p53 Ab DO-1. A representative staining of C91/PL, demonstrating clear nuclear staining for the protein, is shown.
    Figure Legend Snippet: p53 is not transcriptionally functional in HTLV-1-infected cell lines. U2OS cells were transfected by the Ca 2 PO 4 precipitation method with 1 μg of reporter plasmid and 1 μg of TK-renilla-Luc, an internal control plasmid to normalize transfection. Lysates were assayed for firefly luciferase and Renilla luciferase by using a dual assay kit (Promega). HTLV-1-infected cell lines were transfected by the DEAE-Dextran method with 1 μg each of the above-mentioned plasmids. Jurkat cells were transfected with Superfect with the same amounts of DNA as for the other cell lines. Error bars show the standard deviations of four independent experiments and are normalized for protein amount. (A) Fold induction by endogenous p53 activity on the p53-responsive plasmid PG13-Luc. The control plasmid was a construct containing a basal promoter expressing the firefly luciferase gene that contained no inducible element. U2OS expresses wild-type p53 and shows strong activation of PG13-Luc. Jurkat expresses a nonfunctional p53 protein and has no activity on this construct. All four of the HTLV-1-infected cell lines show no activity on PG13-Luc, even though they express high levels of wild-type p53. Both IL-2-dependent and -independent lines are negative for p53 transactivation. (B) Fold induction on the HTLV-1-LTR-Luc construct. This plasmid contains the entire HTLV-1 long terminal repeat cloned upstream of a basal promoter present in the PGL3-Luc construct and is strongly activated by HTLV-1 Tax. All four HTLV-1-infected cell lines activate this reporter, but neither U2OS nor Jurkat transactivate this construct to any extent. (C) Relative luciferase activity on the TK-renilla-Luc construct in the different cell lines. (D) Western blot analysis for the presence of endogenous p53 and Tax. Forty micrograms of protein lysate from luciferase assays was run on SDS–10% PAGE gels, and proteins were detected by chemiluminescence. The HTLV-1-infected cells express at least as much p53 protein as U2OS but show no activity on the PG13-Luc reporter gene (panel A). (E) Cells were mounted on slides by using cytospin funnels and were fixed for 10 min with 2% paraformaldehyde. Indirect immunofluorescence analysis was performed with the anti-p53 Ab DO-1. A representative staining of C91/PL, demonstrating clear nuclear staining for the protein, is shown.

    Techniques Used: Functional Assay, Infection, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Construct, Expressing, Activation Assay, Clone Assay, Western Blot, Polyacrylamide Gel Electrophoresis, Immunofluorescence, Staining

    22) Product Images from "Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors"

    Article Title: Transcriptional repression by AML1 and LEF-1 is mediated by the TLE/Groucho corepressors

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

    doi:

    TLE1 repress TCRα and TCRβ activity in Jurkat cells. Jurkat cells (2.5 × 10 6 ) were transfected by SuperFect for 7–8 hr with wild-type or mutated (marked by × at the top panel) TCRβ, 2 μg ( A ), or TCRα, 1 μg ( B ) reporters, with or without 1 μg of TLE1 expression vector. Data presented are the average of five (TCRβ) and six (TCRα) independent transfections carried out in duplicate. Control values of reporter lacking TCR enhancer (5–8%) were substracted. ( C ) A scheme summarizing the various interactions of AML/CBFα with the adjacently bound factors Ets, Myb, and C/EBP, as well as with the coactivators (arrows) p300/CBP and ALY, and with the negative regulators (bar) Ear 2 and TLE.
    Figure Legend Snippet: TLE1 repress TCRα and TCRβ activity in Jurkat cells. Jurkat cells (2.5 × 10 6 ) were transfected by SuperFect for 7–8 hr with wild-type or mutated (marked by × at the top panel) TCRβ, 2 μg ( A ), or TCRα, 1 μg ( B ) reporters, with or without 1 μg of TLE1 expression vector. Data presented are the average of five (TCRβ) and six (TCRα) independent transfections carried out in duplicate. Control values of reporter lacking TCR enhancer (5–8%) were substracted. ( C ) A scheme summarizing the various interactions of AML/CBFα with the adjacently bound factors Ets, Myb, and C/EBP, as well as with the coactivators (arrows) p300/CBP and ALY, and with the negative regulators (bar) Ear 2 and TLE.

    Techniques Used: Activity Assay, Transfection, Expressing, Plasmid Preparation

    23) Product Images from "Highly Branched Poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) for High Performance Gene Transfection"

    Article Title: Highly Branched Poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) for High Performance Gene Transfection

    Journal: Polymers

    doi: 10.3390/polym9050161

    Transfection efficiency of LC32/DNA, HC32-103/DNA, PEI and SuperFect/DNA polyplexes formulated with different w / w ratios 48 h post-treatment: ( a ) GFP expression of HeLa cells treated with polyplexes; ( b ) Gluciferase activity of HeLa cells after treatment with polyplexes; ( c ) GFP expression of RDEBK cells; ( d ) Gluciferase activity of RDEBK cells after treatment with polyplexes, 40× magnification. Data points marked with an asterisk are statistically significant relative to the PEI and SuperFect group ( p
    Figure Legend Snippet: Transfection efficiency of LC32/DNA, HC32-103/DNA, PEI and SuperFect/DNA polyplexes formulated with different w / w ratios 48 h post-treatment: ( a ) GFP expression of HeLa cells treated with polyplexes; ( b ) Gluciferase activity of HeLa cells after treatment with polyplexes; ( c ) GFP expression of RDEBK cells; ( d ) Gluciferase activity of RDEBK cells after treatment with polyplexes, 40× magnification. Data points marked with an asterisk are statistically significant relative to the PEI and SuperFect group ( p

    Techniques Used: Transfection, Expressing, Activity Assay

    24) Product Images from "Bcl-2 is a monomeric protein: prevention of homodimerization by structural constraints"

    Article Title: Bcl-2 is a monomeric protein: prevention of homodimerization by structural constraints

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.7.1534

    Fig. 7. Dislocation of mitochondria-associated Bcl–2ΔBH1/2, but not Bcl–2ΔBH1/2/3, to the ER/nuclear membrane by Bcl–2 in R6 cells. ( A ) α–mBcl–2/10C4 (fluorescein) and α–Tom20 (Texas Red) fluorescence analysis on R6 cells transfected with mBcl–2ΔBH1/2 or mBcl–2ΔBH1/2/3 at 24 h post-transfection. ( B ) α–mBcl–2/27–6 (fluorescein) and α–hBcl–2/100 (Texas Red) fluorescence analysis on R6 cells co-transfected with mBcl–2ΔBH1/2 and hBcl–2 or mBcl–2ΔBH1/2/3 and hBcl–2 at 24 h post-transfection. In this co-localization experiment α–mBcl–2/10C4 cannot be used because it is a monoclonal mouse antibody like α–hBcl–2/100. However, α–mBcl–2/27–6 and α–mBcl–2/10C4 exhibit the same immunostaining of mBcl–2ΔBH1/2 and mBcl–2ΔBH1/2/3 (our unpublished data). Note that mBcl–2ΔBH1/2 dislocates from its original mitochondrial to a nuclear envelope/ER pattern upon co-transfection with hBcl–2. In contrast, mBcl–2ΔBH1/2/3 does not co-localize with hBcl–2 on the nuclear envelope. The vacuoles are due to the transient transfection with Superfect.
    Figure Legend Snippet: Fig. 7. Dislocation of mitochondria-associated Bcl–2ΔBH1/2, but not Bcl–2ΔBH1/2/3, to the ER/nuclear membrane by Bcl–2 in R6 cells. ( A ) α–mBcl–2/10C4 (fluorescein) and α–Tom20 (Texas Red) fluorescence analysis on R6 cells transfected with mBcl–2ΔBH1/2 or mBcl–2ΔBH1/2/3 at 24 h post-transfection. ( B ) α–mBcl–2/27–6 (fluorescein) and α–hBcl–2/100 (Texas Red) fluorescence analysis on R6 cells co-transfected with mBcl–2ΔBH1/2 and hBcl–2 or mBcl–2ΔBH1/2/3 and hBcl–2 at 24 h post-transfection. In this co-localization experiment α–mBcl–2/10C4 cannot be used because it is a monoclonal mouse antibody like α–hBcl–2/100. However, α–mBcl–2/27–6 and α–mBcl–2/10C4 exhibit the same immunostaining of mBcl–2ΔBH1/2 and mBcl–2ΔBH1/2/3 (our unpublished data). Note that mBcl–2ΔBH1/2 dislocates from its original mitochondrial to a nuclear envelope/ER pattern upon co-transfection with hBcl–2. In contrast, mBcl–2ΔBH1/2/3 does not co-localize with hBcl–2 on the nuclear envelope. The vacuoles are due to the transient transfection with Superfect.

    Techniques Used: Fluorescence, Transfection, Immunostaining, Cotransfection

    Related Articles

    Transfection:

    Article Title: Oxalate-inducible AMBP gene and its regulatory mechanism in renal tubular epithelial cells
    Article Snippet: .. ApH4ON or a mock cis element corresponding to OctON (octamer transcription factor binding cis element oligonucleotide) were transfected using SuperFect™ reagent following the manufacturer's instructions (Qiagen). ..

    Article Title: CREB Activation Induces Adipogenesis in 3T3-L1 Cells
    Article Snippet: .. Plates of 3T3-L1 fibroblasts and adipocytes were grown to 70 to 80% confluency and transfected with the indicated plasmids with Superfect Reagent (Qiagen, Valencia, Calif.) according to the manufacturer's recommendations. .. Cells stably transfected with the plasmid pVgRXR were selected in conventional medium containing 500 μg of Zeocin per ml, and cells stably transfected with pIND-VP16-CREB, pIND-KCREB (or pIND-VP16-KCREB and pIND-LacZ) plasmids were selected in medium containing 500 μg of Geneticin per ml.

    Article Title: FHL2 interacts with and acts as a functional repressor of Id2 in human neuroblastoma cells
    Article Snippet: .. DNA transfection involved use of Superfect reagent (Qiagen, Hilden, Germany). .. Transfection with siRNA duplexes involved use of Lipofectamine 2000 (Invitrogen).

    Article Title: Protein Kinase A Regulates Cholinergic Gene Expression in PC12 Cells: REST4 Silences the Silencing Activity of Neuron-Restrictive Silencer Factor/REST
    Article Snippet: .. SuperFect transfection reagent, plasmid kits, and RNeasy MINI kits (Total RNA system) were obtained from Qiagen Inc. (Valencia, Calif.). .. The GalactoLight system was purchased from Tropix Inc. (Bedford, Mass.).

    Article Title: Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3
    Article Snippet: .. For the transient transfection assay, subconfluent cells (5 × 105 /60-mm dish) were transfected with 1–2 μg of the reporter plasmids and indicated amounts of IRF expression plasmids using Superfect transfection reagent (Qiagen). .. The total amount of DNA used in each transfection was kept constant, and the β-galactosidase expressing plasmid (0.1 μg) was included as internal standard.

    Incubation:

    Article Title: NF-?B Controls Cell Growth and Differentiation through Transcriptional Regulation of Cyclin D1
    Article Snippet: .. The following day, a total of 2.5 μg of plasmid DNA was incubated with Superfect as recommended by the manufacturer (Qiagen). ..

    Expressing:

    Article Title: Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3
    Article Snippet: .. For the transient transfection assay, subconfluent cells (5 × 105 /60-mm dish) were transfected with 1–2 μg of the reporter plasmids and indicated amounts of IRF expression plasmids using Superfect transfection reagent (Qiagen). .. The total amount of DNA used in each transfection was kept constant, and the β-galactosidase expressing plasmid (0.1 μg) was included as internal standard.

    Binding Assay:

    Article Title: Oxalate-inducible AMBP gene and its regulatory mechanism in renal tubular epithelial cells
    Article Snippet: .. ApH4ON or a mock cis element corresponding to OctON (octamer transcription factor binding cis element oligonucleotide) were transfected using SuperFect™ reagent following the manufacturer's instructions (Qiagen). ..

    Transient Transfection Assay:

    Article Title: Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3
    Article Snippet: .. For the transient transfection assay, subconfluent cells (5 × 105 /60-mm dish) were transfected with 1–2 μg of the reporter plasmids and indicated amounts of IRF expression plasmids using Superfect transfection reagent (Qiagen). .. The total amount of DNA used in each transfection was kept constant, and the β-galactosidase expressing plasmid (0.1 μg) was included as internal standard.

    Plasmid Preparation:

    Article Title: Protein Kinase A Regulates Cholinergic Gene Expression in PC12 Cells: REST4 Silences the Silencing Activity of Neuron-Restrictive Silencer Factor/REST
    Article Snippet: .. SuperFect transfection reagent, plasmid kits, and RNeasy MINI kits (Total RNA system) were obtained from Qiagen Inc. (Valencia, Calif.). .. The GalactoLight system was purchased from Tropix Inc. (Bedford, Mass.).

    Article Title: NF-?B Controls Cell Growth and Differentiation through Transcriptional Regulation of Cyclin D1
    Article Snippet: .. The following day, a total of 2.5 μg of plasmid DNA was incubated with Superfect as recommended by the manufacturer (Qiagen). ..

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    FHL2 competitively blunts the binding of Id2 to E47 and releases E47 to its cognate <t>DNA.</t> ( A ) FHL2 blunts the E47–Id2 association in MCF-7 cells. Myc-tagged Id2, E47 and/or Flag-tagged FHL2 were transiently cotransfected into MCF-7 cells. After 48 h <t>transfection,</t> cells were harvested and fractionated to a nuclear fraction. Equal amounts of nuclear lysates were immunoprecipitated and immunoblotted with the indicated antibodies. ( B–D ) FHL2 attenuates Id-inhibited E47–DNA binding in vitro . EMSA was performed as described in Materials and Methods section. ( E ) Knock down of the endogenous FHL2 in MCF-7 cells sharply attenuated the binding of the endogenous E47 to the exogenous target DNA sequence. ChIP assays were performed as described in Materials and methods section. NS, non-specific band.
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    FHL2 competitively blunts the binding of Id2 to E47 and releases E47 to its cognate DNA. ( A ) FHL2 blunts the E47–Id2 association in MCF-7 cells. Myc-tagged Id2, E47 and/or Flag-tagged FHL2 were transiently cotransfected into MCF-7 cells. After 48 h transfection, cells were harvested and fractionated to a nuclear fraction. Equal amounts of nuclear lysates were immunoprecipitated and immunoblotted with the indicated antibodies. ( B–D ) FHL2 attenuates Id-inhibited E47–DNA binding in vitro . EMSA was performed as described in Materials and Methods section. ( E ) Knock down of the endogenous FHL2 in MCF-7 cells sharply attenuated the binding of the endogenous E47 to the exogenous target DNA sequence. ChIP assays were performed as described in Materials and methods section. NS, non-specific band.

    Journal: Nucleic Acids Research

    Article Title: FHL2 interacts with and acts as a functional repressor of Id2 in human neuroblastoma cells

    doi: 10.1093/nar/gkp332

    Figure Lengend Snippet: FHL2 competitively blunts the binding of Id2 to E47 and releases E47 to its cognate DNA. ( A ) FHL2 blunts the E47–Id2 association in MCF-7 cells. Myc-tagged Id2, E47 and/or Flag-tagged FHL2 were transiently cotransfected into MCF-7 cells. After 48 h transfection, cells were harvested and fractionated to a nuclear fraction. Equal amounts of nuclear lysates were immunoprecipitated and immunoblotted with the indicated antibodies. ( B–D ) FHL2 attenuates Id-inhibited E47–DNA binding in vitro . EMSA was performed as described in Materials and Methods section. ( E ) Knock down of the endogenous FHL2 in MCF-7 cells sharply attenuated the binding of the endogenous E47 to the exogenous target DNA sequence. ChIP assays were performed as described in Materials and methods section. NS, non-specific band.

    Article Snippet: DNA transfection involved use of Superfect reagent (Qiagen, Hilden, Germany).

    Techniques: Binding Assay, Transfection, Immunoprecipitation, In Vitro, Sequencing, Chromatin Immunoprecipitation

    Overexpression of IRF-3 enhanced the NDV-mediated induction of endogenous IFNA4 and IFNB in REF cells. ( A ). The data represent the results from three independent transfection experiments and assays. ( B ) RT-PCR. REF cells were transiently transfected with 4 μg of either pSp64 (lanes 1 and 3) or IRF-3 (lanes 2 and 4) by Superfect (Qiagen) transfection reagent. Two micrograms of purified RNA was amplified by RT-PCR method with specific primers for rat IFNA as described in Materials and Methods . Amplified fragments were separated on an agarose gel and visualized by ethidium bromide staining (Lt), or transferred onto nitrocellulose, and hybridized with the IFNA-specific riboprobe (Rt). ( C ) REF cells were transiently transfected with a control or IRF-3 plasmid. Twenty-four hours later, cells were infected for the time periods indicated. RNA was collected and analyzed by Northern hybridization to an IFNB-specific riboprobe.

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

    Article Title: Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3

    doi:

    Figure Lengend Snippet: Overexpression of IRF-3 enhanced the NDV-mediated induction of endogenous IFNA4 and IFNB in REF cells. ( A ). The data represent the results from three independent transfection experiments and assays. ( B ) RT-PCR. REF cells were transiently transfected with 4 μg of either pSp64 (lanes 1 and 3) or IRF-3 (lanes 2 and 4) by Superfect (Qiagen) transfection reagent. Two micrograms of purified RNA was amplified by RT-PCR method with specific primers for rat IFNA as described in Materials and Methods . Amplified fragments were separated on an agarose gel and visualized by ethidium bromide staining (Lt), or transferred onto nitrocellulose, and hybridized with the IFNA-specific riboprobe (Rt). ( C ) REF cells were transiently transfected with a control or IRF-3 plasmid. Twenty-four hours later, cells were infected for the time periods indicated. RNA was collected and analyzed by Northern hybridization to an IFNB-specific riboprobe.

    Article Snippet: For the transient transfection assay, subconfluent cells (5 × 105 /60-mm dish) were transfected with 1–2 μg of the reporter plasmids and indicated amounts of IRF expression plasmids using Superfect transfection reagent (Qiagen).

    Techniques: Over Expression, Transfection, Reverse Transcription Polymerase Chain Reaction, Purification, Amplification, Agarose Gel Electrophoresis, Staining, Plasmid Preparation, Infection, Northern Blot, Hybridization

    ChAT activity and ChAT and VAChT mRNAs in A126.1B2 cells transfected with REST4. (A) A126.1B2 was transfected with pCS2+MT-REST4 (+REST4) or vector alone (−REST4), using SuperFect transfection reagent. After 72 h, cell extracts were prepared and assayed for REST4 expression by Western blot analysis (top) or for ChAT activity (bottom) as described in Materials and Methods. (B) PCR analysis for ChAT mRNA (30 cycles) and VAChT mRNA (35 cycles) of A126.1B2 treated as described above.

    Journal: Molecular and Cellular Biology

    Article Title: Protein Kinase A Regulates Cholinergic Gene Expression in PC12 Cells: REST4 Silences the Silencing Activity of Neuron-Restrictive Silencer Factor/REST

    doi:

    Figure Lengend Snippet: ChAT activity and ChAT and VAChT mRNAs in A126.1B2 cells transfected with REST4. (A) A126.1B2 was transfected with pCS2+MT-REST4 (+REST4) or vector alone (−REST4), using SuperFect transfection reagent. After 72 h, cell extracts were prepared and assayed for REST4 expression by Western blot analysis (top) or for ChAT activity (bottom) as described in Materials and Methods. (B) PCR analysis for ChAT mRNA (30 cycles) and VAChT mRNA (35 cycles) of A126.1B2 treated as described above.

    Article Snippet: SuperFect transfection reagent, plasmid kits, and RNeasy MINI kits (Total RNA system) were obtained from Qiagen Inc. (Valencia, Calif.).

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Polymerase Chain Reaction

    CREB is expressed before and during adipogenesis, and differentiation-inducing agents stimulate CREB phoshorylation and transcriptional activity. (A) 3T3-L1 preadipocytes were grown to confluency as described in Materials and Methods. The cells were refed with complete growth medium containing 1 μg of insulin per ml, 1 μM dexamethasone, and 0.3 mM Bt 2 cAMP for the times indicated above each lane. Approximately 25 μg of cell lysate protein from each sample was separated on 10% acrylamide–SDS gels and transferred to nitrocellulose membranes. Duplicate membranes were subjected to Western analysis by using antibodies specific for Ser133 phosphorylated CREB (Phospho-CREB), total CREB, or CEBPβ protein as indicated. (B) Preadipocytes were grown to confluency and then refed with medium containing insulin, dexamethasone, and Bt 2 cAMP for 48 h. The cells were then refed every 2 days with medium containing 1 μg of insulin per ml. Cell lysates were prepared on the days indicated above each lane, and 25 μg of lysate protein from each sample was separated on 10% polyacrylamide–SDS gels and transferred to nitrocellulose membranes. Individual membranes were probed with antibodies specific for phospho-CREB, total CREB, CEBPα and -β, and RXRα as indicated. (C) 3T3-L1 fibroblasts and adipocytes were grown as described in Materials and Methods. Cells were transfected with the plasmid, pGal4TK-Luc, alone or with the plasmid pRSV-Gal4-CREB by using Superfect transfection reagent. The plasmids are described in the main text. At 24 h after transfection, the cells were treated with 0.5 mM Bt 2 cAMP alone or with a mixture of 1 μg of insulin per ml, 1 μM dexamethasone, and 0.5 mM Bt 2 cAMP for 4 h. The control cells received no treatment. Luciferase levels were measured in cell lysates as an index of transcription from the Gal4-TK promoter. Levels of transcription are shown relative to levels measured in untreated control cells transfected with pGal4TK-Luc alone.

    Journal: Molecular and Cellular Biology

    Article Title: CREB Activation Induces Adipogenesis in 3T3-L1 Cells

    doi:

    Figure Lengend Snippet: CREB is expressed before and during adipogenesis, and differentiation-inducing agents stimulate CREB phoshorylation and transcriptional activity. (A) 3T3-L1 preadipocytes were grown to confluency as described in Materials and Methods. The cells were refed with complete growth medium containing 1 μg of insulin per ml, 1 μM dexamethasone, and 0.3 mM Bt 2 cAMP for the times indicated above each lane. Approximately 25 μg of cell lysate protein from each sample was separated on 10% acrylamide–SDS gels and transferred to nitrocellulose membranes. Duplicate membranes were subjected to Western analysis by using antibodies specific for Ser133 phosphorylated CREB (Phospho-CREB), total CREB, or CEBPβ protein as indicated. (B) Preadipocytes were grown to confluency and then refed with medium containing insulin, dexamethasone, and Bt 2 cAMP for 48 h. The cells were then refed every 2 days with medium containing 1 μg of insulin per ml. Cell lysates were prepared on the days indicated above each lane, and 25 μg of lysate protein from each sample was separated on 10% polyacrylamide–SDS gels and transferred to nitrocellulose membranes. Individual membranes were probed with antibodies specific for phospho-CREB, total CREB, CEBPα and -β, and RXRα as indicated. (C) 3T3-L1 fibroblasts and adipocytes were grown as described in Materials and Methods. Cells were transfected with the plasmid, pGal4TK-Luc, alone or with the plasmid pRSV-Gal4-CREB by using Superfect transfection reagent. The plasmids are described in the main text. At 24 h after transfection, the cells were treated with 0.5 mM Bt 2 cAMP alone or with a mixture of 1 μg of insulin per ml, 1 μM dexamethasone, and 0.5 mM Bt 2 cAMP for 4 h. The control cells received no treatment. Luciferase levels were measured in cell lysates as an index of transcription from the Gal4-TK promoter. Levels of transcription are shown relative to levels measured in untreated control cells transfected with pGal4TK-Luc alone.

    Article Snippet: Plates of 3T3-L1 fibroblasts and adipocytes were grown to 70 to 80% confluency and transfected with the indicated plasmids with Superfect Reagent (Qiagen, Valencia, Calif.) according to the manufacturer's recommendations.

    Techniques: Activity Assay, Western Blot, Transfection, Plasmid Preparation, Luciferase

    Effect of VP16-CREB or KCREB on CRE-dependent gene transcription and 3T3-L1 proliferation. (A) Stably transfected 3T3-L1 clonal cell lines inducibly expressing VP16-CREB (clones 2-4 and 9-7) or KCREB (clones 2-1 and 2-10) were transfected with the plasmid, −109 pPC-Luc, which contains a CREB-responsive portion of PEPCK gene promoter linked to a luciferase reporter gene by using Superfect reagent. The cells were cotransfected with the plasmid pSV-βGal. The following day the cells were treated with 0.3 mM Bt 2 cAMP, 10 μM muristerone, or both agents together as indicated. After 4 h cell lysates were prepared, and the luciferase activity was measured as an index of transcriptional activity. Levels are shown relative to the levels of luciferase activity in untreated, control cells (No Add'n). (B) 3T3-L1 cells stably transfected with pIND-KCREB (clone 2-10) were stimulated with the indicated final concentrations of muristerone for 20 h. Nuclear extracts were prepared, and electrophoretic mobility shift assays were performed with a 32 P-labeled oligonucleotide containing the consensus CRE sequence (TGACGTCA). Reactions were separated on nondenaturing 6% polyacrylamide gels and exposed to film. The figure shows a representative autoradiograph. (C) Stably transfected 3T3-L1 clonal cell lines inducibly expressing VP16-CREB (clones 2-4 and 9-7) or KCREB (clones 2-1 and 2-10) were transferred to duplicate wells of 96-well plates (5,000 cells/well). After 24 h, one well was treated with 10 μg of muristerone (squares, solid line), and the remaining well was left untreated (circles, dotted line). Cell numbers were determined in each well with the Cell Titer 96 AQ reagent system at 24, 48, 72, and 96 h after plating of the cells. Values are shown relative to levels measured in wells containing untreated, control cells at the 24-h time point for each cell line and are the averages of three assays.

    Journal: Molecular and Cellular Biology

    Article Title: CREB Activation Induces Adipogenesis in 3T3-L1 Cells

    doi:

    Figure Lengend Snippet: Effect of VP16-CREB or KCREB on CRE-dependent gene transcription and 3T3-L1 proliferation. (A) Stably transfected 3T3-L1 clonal cell lines inducibly expressing VP16-CREB (clones 2-4 and 9-7) or KCREB (clones 2-1 and 2-10) were transfected with the plasmid, −109 pPC-Luc, which contains a CREB-responsive portion of PEPCK gene promoter linked to a luciferase reporter gene by using Superfect reagent. The cells were cotransfected with the plasmid pSV-βGal. The following day the cells were treated with 0.3 mM Bt 2 cAMP, 10 μM muristerone, or both agents together as indicated. After 4 h cell lysates were prepared, and the luciferase activity was measured as an index of transcriptional activity. Levels are shown relative to the levels of luciferase activity in untreated, control cells (No Add'n). (B) 3T3-L1 cells stably transfected with pIND-KCREB (clone 2-10) were stimulated with the indicated final concentrations of muristerone for 20 h. Nuclear extracts were prepared, and electrophoretic mobility shift assays were performed with a 32 P-labeled oligonucleotide containing the consensus CRE sequence (TGACGTCA). Reactions were separated on nondenaturing 6% polyacrylamide gels and exposed to film. The figure shows a representative autoradiograph. (C) Stably transfected 3T3-L1 clonal cell lines inducibly expressing VP16-CREB (clones 2-4 and 9-7) or KCREB (clones 2-1 and 2-10) were transferred to duplicate wells of 96-well plates (5,000 cells/well). After 24 h, one well was treated with 10 μg of muristerone (squares, solid line), and the remaining well was left untreated (circles, dotted line). Cell numbers were determined in each well with the Cell Titer 96 AQ reagent system at 24, 48, 72, and 96 h after plating of the cells. Values are shown relative to levels measured in wells containing untreated, control cells at the 24-h time point for each cell line and are the averages of three assays.

    Article Snippet: Plates of 3T3-L1 fibroblasts and adipocytes were grown to 70 to 80% confluency and transfected with the indicated plasmids with Superfect Reagent (Qiagen, Valencia, Calif.) according to the manufacturer's recommendations.

    Techniques: Stable Transfection, Transfection, Expressing, Plasmid Preparation, Luciferase, Activity Assay, Electrophoretic Mobility Shift Assay, Labeling, Sequencing, Autoradiography