Structured Review

Qiagen transfection complexes
Exosome concentration affects electroporation efficiency. To determine the optimal exosome concentration for <t>transfection,</t> exosomes at four different concentrations (0.25–4 µg/µl) were mixed with an Alexa Fluor 488-tagged siRNA (2 nmol/ml) and electroporated. Samples containing exosomes and siRNA, without an electric pulse, were used as a negative control. To eliminate the excess siRNA, the exosomes were purified using latex beads and the presence of siRNA in the exosomes was determined by FACS analysis.
Transfection Complexes, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes"

Article Title: Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks463

Exosome concentration affects electroporation efficiency. To determine the optimal exosome concentration for transfection, exosomes at four different concentrations (0.25–4 µg/µl) were mixed with an Alexa Fluor 488-tagged siRNA (2 nmol/ml) and electroporated. Samples containing exosomes and siRNA, without an electric pulse, were used as a negative control. To eliminate the excess siRNA, the exosomes were purified using latex beads and the presence of siRNA in the exosomes was determined by FACS analysis.
Figure Legend Snippet: Exosome concentration affects electroporation efficiency. To determine the optimal exosome concentration for transfection, exosomes at four different concentrations (0.25–4 µg/µl) were mixed with an Alexa Fluor 488-tagged siRNA (2 nmol/ml) and electroporated. Samples containing exosomes and siRNA, without an electric pulse, were used as a negative control. To eliminate the excess siRNA, the exosomes were purified using latex beads and the presence of siRNA in the exosomes was determined by FACS analysis.

Techniques Used: Concentration Assay, Electroporation, Transfection, Negative Control, Purification, FACS

Flow cytometry and northern slot blot of exosomes following chemical transfection. siRNA was embedded into lipid micelles according to the manufacturer’s instructions. The embedded siRNA was mixed with exosomes and incubated overnight. To eliminate the excess siRNA embedded in transfection complexes, the exosomes were purified using latex beads. The beads were washed twice with PBS to eliminate the excess of un-specific bound siRNA molecules. The presence of fluorescently labeled siRNA was determined by flow cytometry and northern blotting (slot blot). Panel A–D shows the results of flow cytometry for the detection of Alexa Fluor-488 labeled siRNA, beads only ( A ), beads plus siRNA ( B ), beads plus siRNA embedded in lipid micelles (negative control; C ) and chemically transfected exosomes ( D ). Panels E and F show the results of northern blotting using a digoxigenin-labeled probe against the administered MAPK1 siRNA. Panel E shows the presence of siRNA in exosomes and micelles. The chemically transfected exosomes were mixed with their parental cells HTB-177 and the delivery of siRNA from the exosomes to cells was determined and compared with direct transfection of cells ( F ). Non-transfected exosomes and cells were used as negative control.
Figure Legend Snippet: Flow cytometry and northern slot blot of exosomes following chemical transfection. siRNA was embedded into lipid micelles according to the manufacturer’s instructions. The embedded siRNA was mixed with exosomes and incubated overnight. To eliminate the excess siRNA embedded in transfection complexes, the exosomes were purified using latex beads. The beads were washed twice with PBS to eliminate the excess of un-specific bound siRNA molecules. The presence of fluorescently labeled siRNA was determined by flow cytometry and northern blotting (slot blot). Panel A–D shows the results of flow cytometry for the detection of Alexa Fluor-488 labeled siRNA, beads only ( A ), beads plus siRNA ( B ), beads plus siRNA embedded in lipid micelles (negative control; C ) and chemically transfected exosomes ( D ). Panels E and F show the results of northern blotting using a digoxigenin-labeled probe against the administered MAPK1 siRNA. Panel E shows the presence of siRNA in exosomes and micelles. The chemically transfected exosomes were mixed with their parental cells HTB-177 and the delivery of siRNA from the exosomes to cells was determined and compared with direct transfection of cells ( F ). Non-transfected exosomes and cells were used as negative control.

Techniques Used: Flow Cytometry, Cytometry, Northern Blot, Dot Blot, Transfection, Incubation, Purification, Labeling, Negative Control

2) Product Images from "Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy"

Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.09-4171

GAPDH in retinal endothelial cells. Retinal endothelial cells were transfected using 0.5 to 5.0 μg GAPDH expression plasmid for 8 hours. ( a ) GAPDH activity was measured spectrophotometrically to determine the degree of transfection. ( b , c ) BRECs were transfected with 2.5 μg GAPDH plasmid or no plasmid but with transfection reagent alone (R) for 8 hours and were incubated for 4 days in 5 mM or 20 mM glucose media. ( b ) GAPDH abundance was assessed by Western blot analysis, and ( c ) its glycolytic activity was assessed by measuring the production of NADH at 340 nm. Each parameter was measured in duplicate using four different cell preparations. Results are presented as mean ± SD of the values obtained from three different cell preparations, with each measurement made in duplicate. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected BRECs incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone, followed by incubation in 5 mM or 20 mM glucose, respectively. * P
Figure Legend Snippet: GAPDH in retinal endothelial cells. Retinal endothelial cells were transfected using 0.5 to 5.0 μg GAPDH expression plasmid for 8 hours. ( a ) GAPDH activity was measured spectrophotometrically to determine the degree of transfection. ( b , c ) BRECs were transfected with 2.5 μg GAPDH plasmid or no plasmid but with transfection reagent alone (R) for 8 hours and were incubated for 4 days in 5 mM or 20 mM glucose media. ( b ) GAPDH abundance was assessed by Western blot analysis, and ( c ) its glycolytic activity was assessed by measuring the production of NADH at 340 nm. Each parameter was measured in duplicate using four different cell preparations. Results are presented as mean ± SD of the values obtained from three different cell preparations, with each measurement made in duplicate. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected BRECs incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone, followed by incubation in 5 mM or 20 mM glucose, respectively. * P

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

Effect of GAPDH overexpression on endothelial cell death: GAPDH-transfected BRECs (using 2.5 μg plasmid) or untransfected BRECs were incubated in 5 mM or 20 mM glucose for 4 days. ( a ) Apoptosis was determined by cytoplasmic histone-associated DNA fragments using ELISA. The graph shows the mean ± SD adjusted to the total DNA in each sample. ( b ) Activation of apoptosis execution enzyme caspase-3 was determined in the cells by measuring the cleavage of the substrate Ac-DEVD-pNA. Each experiment was repeated with at least three different BREC preparations, and measurements were made in duplicate. ( c ) The appearance of a 85-kDa band of PARP was determined by Western blot technique, and β-actin was used as a loading standard. Western blot is representative of four different experiments. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 20+Fe, 20 mM glucose and 2.5 μM FeTPPS; 20+PJ, 20 mM glucose and 1.0 μM PJ34; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone before incubation with 5 mM or 20 mM glucose, respectively. The values, represented as mean ± SD, obtained with 5 mM glucose were considered 100%. * P
Figure Legend Snippet: Effect of GAPDH overexpression on endothelial cell death: GAPDH-transfected BRECs (using 2.5 μg plasmid) or untransfected BRECs were incubated in 5 mM or 20 mM glucose for 4 days. ( a ) Apoptosis was determined by cytoplasmic histone-associated DNA fragments using ELISA. The graph shows the mean ± SD adjusted to the total DNA in each sample. ( b ) Activation of apoptosis execution enzyme caspase-3 was determined in the cells by measuring the cleavage of the substrate Ac-DEVD-pNA. Each experiment was repeated with at least three different BREC preparations, and measurements were made in duplicate. ( c ) The appearance of a 85-kDa band of PARP was determined by Western blot technique, and β-actin was used as a loading standard. Western blot is representative of four different experiments. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 20+Fe, 20 mM glucose and 2.5 μM FeTPPS; 20+PJ, 20 mM glucose and 1.0 μM PJ34; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone before incubation with 5 mM or 20 mM glucose, respectively. The values, represented as mean ± SD, obtained with 5 mM glucose were considered 100%. * P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

Subcellular distribution of GAPDH. BRECs either transfected with 2.5 μg GAPDH plasmid or left untransfected were incubated for 4 days in 5 mM or 20 mM glucose media. The nuclear fraction was prepared by centrifugation, and the expression of GAPDH was determined by Western blot analysis using histone 2B as a loading control. Western blot represents results obtained from three or four different experiments, with each measurement made in duplicate. Histogram represents mean ± SD. GAPDH band intensity adjusted to H2B band intensity, and the values obtained from BRECs incubated in 5 mM glucose are considered 100%. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 20+R, BRECs treated with the transfection reagent alone before incubation with 20 mM glucose. * P
Figure Legend Snippet: Subcellular distribution of GAPDH. BRECs either transfected with 2.5 μg GAPDH plasmid or left untransfected were incubated for 4 days in 5 mM or 20 mM glucose media. The nuclear fraction was prepared by centrifugation, and the expression of GAPDH was determined by Western blot analysis using histone 2B as a loading control. Western blot represents results obtained from three or four different experiments, with each measurement made in duplicate. Histogram represents mean ± SD. GAPDH band intensity adjusted to H2B band intensity, and the values obtained from BRECs incubated in 5 mM glucose are considered 100%. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 20+R, BRECs treated with the transfection reagent alone before incubation with 20 mM glucose. * P

Techniques Used: Transfection, Plasmid Preparation, Incubation, Centrifugation, Expressing, Western Blot

3) Product Images from "IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling"

Article Title: IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling

Journal: PLoS ONE

doi: 10.1371/journal.pone.0156002

Stress fiber formation in IL-4 treated HCAEC. Live actin-RFP staining showing the formation of actin stress fibers in HCAEC treated with IL-4 or Wnt5A in the absence or presence of sFRP1 and WIF1. Expression of de novo synthesized RFP-actin after transfection as outlined in methods was visually observed up to 24 h. Photomicrographs of stress fiber formation were taken randomly at 12 h after stimulation with IL-4 and Wnt5A alone or in the presence of sFRP1 and WIF1 using Zeiss Axio Observer.Z1 equipped with AxioCam MRm digital camera and ZEN 2012 software. Original magnification, 400×. Independent identical experiments in triplicates were repeated at least three times, with analogous results.
Figure Legend Snippet: Stress fiber formation in IL-4 treated HCAEC. Live actin-RFP staining showing the formation of actin stress fibers in HCAEC treated with IL-4 or Wnt5A in the absence or presence of sFRP1 and WIF1. Expression of de novo synthesized RFP-actin after transfection as outlined in methods was visually observed up to 24 h. Photomicrographs of stress fiber formation were taken randomly at 12 h after stimulation with IL-4 and Wnt5A alone or in the presence of sFRP1 and WIF1 using Zeiss Axio Observer.Z1 equipped with AxioCam MRm digital camera and ZEN 2012 software. Original magnification, 400×. Independent identical experiments in triplicates were repeated at least three times, with analogous results.

Techniques Used: Staining, Expressing, Synthesized, Transfection, Software

4) Product Images from "Oxidative damage of mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase"

Article Title: Oxidative damage of mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase

Journal: Free radical research

doi: 10.3109/10715760903494168

Effect of glucose on OGG1 and protection by MnSOD:RNA isolated from the cells treated with MnTBAP or transfected with MnSOD or treated with the transfection reagent alone and was assessed by real-time RT-PCR for OGG1. The values were normalized to 18 s mRNA in each sample. Fold-change relative to the values obtained from un-transfected cells incubated in 5 mM glucose was calculated using the ddCt method. Results are from the measurements made in three-to-five preparations. * p
Figure Legend Snippet: Effect of glucose on OGG1 and protection by MnSOD:RNA isolated from the cells treated with MnTBAP or transfected with MnSOD or treated with the transfection reagent alone and was assessed by real-time RT-PCR for OGG1. The values were normalized to 18 s mRNA in each sample. Fold-change relative to the values obtained from un-transfected cells incubated in 5 mM glucose was calculated using the ddCt method. Results are from the measurements made in three-to-five preparations. * p

Techniques Used: Isolation, Transfection, Quantitative RT-PCR, Incubation

Effect of high glucose on mitochondrial DNA damage: DNA damage was assessed using 15 ng total DNA and mitochondrial or nuclear specific primers for long and short PCR products in the cells incubated with 5 mM glucose or 20 mM glucose in the presence of MnTBAP or from the cells transfected with MnSOD or Mock cells incubated in 5 mM or 20 mM glucose media. (A) Relative amplification was calculated by normalizing the intensity of the 13.3 kb product to the 232 bp product for mtDNA and (B) the 13.8 kb product to the 241 bp product for nDNA. Each measurement was made in duplicate in at least three different cell preparations. 5=5 mM glucose, 20=20 mM glucose, 20+MnTb=20 mM glucose + 200 μM MnTBAP, SOD=cells transfected with MnSOD, Mock=cells treated with the transfection reagent alone. * p
Figure Legend Snippet: Effect of high glucose on mitochondrial DNA damage: DNA damage was assessed using 15 ng total DNA and mitochondrial or nuclear specific primers for long and short PCR products in the cells incubated with 5 mM glucose or 20 mM glucose in the presence of MnTBAP or from the cells transfected with MnSOD or Mock cells incubated in 5 mM or 20 mM glucose media. (A) Relative amplification was calculated by normalizing the intensity of the 13.3 kb product to the 232 bp product for mtDNA and (B) the 13.8 kb product to the 241 bp product for nDNA. Each measurement was made in duplicate in at least three different cell preparations. 5=5 mM glucose, 20=20 mM glucose, 20+MnTb=20 mM glucose + 200 μM MnTBAP, SOD=cells transfected with MnSOD, Mock=cells treated with the transfection reagent alone. * p

Techniques Used: Polymerase Chain Reaction, Incubation, Transfection, Amplification

5) Product Images from "Expression and regulatory effects of microRNA-182 in osteosarcoma cells: A pilot study"

Article Title: Expression and regulatory effects of microRNA-182 in osteosarcoma cells: A pilot study

Journal: Oncology Letters

doi: 10.3892/ol.2016.4375

Effects on miRNA-182 expression following transfection of human osteosarcoma MG-63 cells with a miRNA-182 inhibitor or mimic. The expression levels of miRNA-182 in the miRNA-182 inhibitor transfection group were significantly decreased compared with the control groups. The expression level of miRNA-182 in the cells transfected with miRNA-182 mimic was significantly increased compared with the control groups. Experiments were repeated three times. Data are presented as the mean ± standard deviation. **P
Figure Legend Snippet: Effects on miRNA-182 expression following transfection of human osteosarcoma MG-63 cells with a miRNA-182 inhibitor or mimic. The expression levels of miRNA-182 in the miRNA-182 inhibitor transfection group were significantly decreased compared with the control groups. The expression level of miRNA-182 in the cells transfected with miRNA-182 mimic was significantly increased compared with the control groups. Experiments were repeated three times. Data are presented as the mean ± standard deviation. **P

Techniques Used: Expressing, Transfection, Standard Deviation

Transfection efficiency. Fluorescence microscopy of human osteosarcoma MG-63 cells transfected with the fluorescence control demonstrated that transfection efficiency was high (magnification, ×100).
Figure Legend Snippet: Transfection efficiency. Fluorescence microscopy of human osteosarcoma MG-63 cells transfected with the fluorescence control demonstrated that transfection efficiency was high (magnification, ×100).

Techniques Used: Transfection, Fluorescence, Microscopy

6) Product Images from "High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity"

Article Title: High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkg884

Activity of the ERCC1 promoter is affected by HMGA2, HMGA2/LPP and ΔHMGA2. ERCC1 promoter fragments –3900 to +1 and –425 to +1 relative to the transcriptional start site were cloned in luciferase reporter-gene vector pGL3-Basic. These promoter constructs were transiently co-transfected with a vector expressing either no HMGA2 (reference vector), wild-type HMGA2, chimeric HMGA2/LPP or C-terminally truncated ΔHMGA2 and vector pRL-Tk were used for normalization. Results are the mean value of several independent experiments performed within HeLa cells. The change in promoter activity of the 3.9 kb and 426 bp ERCC1 promoter fragments in dependence of different HMGA2 protein variants are shown relative to the data obtained for the corresponding ERCC1 construct co-transfected with the reference vector. Black, white and gray bars refer to co-transfection with wild-type HMGA2, chimeric HMGA2/LPP and truncated ΔHMG2, respectively.
Figure Legend Snippet: Activity of the ERCC1 promoter is affected by HMGA2, HMGA2/LPP and ΔHMGA2. ERCC1 promoter fragments –3900 to +1 and –425 to +1 relative to the transcriptional start site were cloned in luciferase reporter-gene vector pGL3-Basic. These promoter constructs were transiently co-transfected with a vector expressing either no HMGA2 (reference vector), wild-type HMGA2, chimeric HMGA2/LPP or C-terminally truncated ΔHMGA2 and vector pRL-Tk were used for normalization. Results are the mean value of several independent experiments performed within HeLa cells. The change in promoter activity of the 3.9 kb and 426 bp ERCC1 promoter fragments in dependence of different HMGA2 protein variants are shown relative to the data obtained for the corresponding ERCC1 construct co-transfected with the reference vector. Black, white and gray bars refer to co-transfection with wild-type HMGA2, chimeric HMGA2/LPP and truncated ΔHMG2, respectively.

Techniques Used: Activity Assay, Clone Assay, Luciferase, Plasmid Preparation, Construct, Transfection, Expressing, Cotransfection

Related Articles

Transfection:

Article Title: High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity
Article Snippet: .. Transfection complexes containing 1 µg of promoter construct DNA, 1 µg of HMGA2 expression plasmid, 250 ng of pRL-TK and 10 µl of SuperFect transfection reagent (Qiagen, Hilden, Germany) were formed in a total volume of 100 µl in TC199 medium (without supplements) by incubating the sample for 10 min at room temperature according to the instructions of the manufacturer. .. After an incubation for 3 h, cells were washed with PBS, 2.5 ml of fresh 20% culture medium was added and cells were then grown for a further 48 h with renewal of the growth medium after 24 h. Luciferase activities were measured in a luminometer, (Biocounter M2010, Lumac BV, The Netherlands) using the Dual-Luciferase Reporter Assay System (Promega) following the instructions of the manufacturer.

Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy
Article Snippet: .. Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days. .. Parallel incubations were carried out by incubating the cells in the transfection reagent alone for 8 hours followed by incubation in 5 mM or 20 mM glucose media. (Our previous studies have shown that control plasmid vectors [pGL3] or scrambled siRNA have no effect on retinal endothelial cells , ).

Article Title: Arsenic trioxide and auranofin inhibit selenoprotein synthesis: implications for chemotherapy for acute promyelocytic leukaemia
Article Snippet: .. Transfection complexes were prepared by a mixture of serum-free media, HiPerfect transfection reagent (Qiagen) and 5 n M siRNA. .. These reagents were incubated for 10 min at room temperature to allow transfection complexes to form and were subsequently added dropwise to cells for optimal transfection efficiency.

Article Title: IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling
Article Snippet: .. Wnt5A Silencing To knockdown Wnt5A expression, HCAEC grown up to 80% confluency in 24-well plates were incubated with transfection complexes formed from Wnt5A-siRNA (5 nM; Hs_Wnt5A_6, Cat. No. SI03025596; Qiagen) and 6 μL/mL HiPerFect Transfection reagent (Qiagen GmbH, Hilden, Germany) in EBM-2 basal medium (Clonetics, Lonza, USA) for 24 h. After splitting using Trypsin EDTA, transfected cells were seeded onto collagen coated 6 well plates, retransfected for an additional round as above and incubated until the cells reached 90% confluency. .. HCAEC were also transfected with validated AllStars negative control siRNA (Qiagen) in parallel to control for ‘off target’ effects of siRNAs.

Article Title: Expression and regulatory effects of microRNA-182 in osteosarcoma cells: A pilot study
Article Snippet: .. Following aspiration, transfection complexes were formed by mixing 250 µl culture medium without FBS, 10 µl HiPerFect® Transfection Reagent (Qiagen, Inc., Valencia, CA, USA) and 10 µl miRNA-182-5p inhibitor (100 nM; catalog no., miR20000259-1-5; Guangzhou RiboBio Co., Ltd., Guangzhou, China) or 8 µl miRNA-182-5p mimic (80 nM; catalog no., miR10000259-1-5; Guangzhou RiboBio Co., Ltd.). .. Subsequently, the mixtures were added dropwise to the cells, and the plates were gently swirled to ensure uniform distribution of the transfection complexes.

Article Title: Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes
Article Snippet: .. To examine the efficiency of chemical transfection, a siRNA against MAPK1 was mixed with transfection reagent to allow for the formation of transfection complexes (siRNA embedded in lipid micelles), according to the manufacturer’s instructions (RNAi Human/Mouse Starter Kit, Quiagen). .. The complex was then added to the exosomes (1 µg/µl) and incubated overnight.

Article Title: Mutations Affecting the BHLHA9 DNA-Binding Domain Cause MSSD, Mesoaxial Synostotic Syndactyly with Phalangeal Reduction, Malik-Percin Type
Article Snippet: .. After 24 hr, for each of the wells, transfection complexes composed of 3 ml Attractene (QIAGEN) and 500 ng DNA of recombinant constructs expressing human wild-type or mutant HsBHLHA9_MYC/DDK and/or HsTCF4_GFP fusion proteins were mixed in 120 μl DMEM without serum, incubated for 20 min at room temperature to allow formation of transfection complexes, and subsequently added drop wise to the cells in 2 ml fresh DMEM_10%. .. The cells with the transfection complexes were incubated under normal growth conditions.

Article Title: Oxidative damage of mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase
Article Snippet: .. Briefly, transfection complexes were formed using Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions and incubated for 8 h The cells were rinsed with DMEM and incubated in 5 mM (normal) or 20 mM (high) glucose media. .. Parallel incubations were carried out using the transfection reagent alone (Mock).

Mutagenesis:

Article Title: Mutations Affecting the BHLHA9 DNA-Binding Domain Cause MSSD, Mesoaxial Synostotic Syndactyly with Phalangeal Reduction, Malik-Percin Type
Article Snippet: .. After 24 hr, for each of the wells, transfection complexes composed of 3 ml Attractene (QIAGEN) and 500 ng DNA of recombinant constructs expressing human wild-type or mutant HsBHLHA9_MYC/DDK and/or HsTCF4_GFP fusion proteins were mixed in 120 μl DMEM without serum, incubated for 20 min at room temperature to allow formation of transfection complexes, and subsequently added drop wise to the cells in 2 ml fresh DMEM_10%. .. The cells with the transfection complexes were incubated under normal growth conditions.

Construct:

Article Title: High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity
Article Snippet: .. Transfection complexes containing 1 µg of promoter construct DNA, 1 µg of HMGA2 expression plasmid, 250 ng of pRL-TK and 10 µl of SuperFect transfection reagent (Qiagen, Hilden, Germany) were formed in a total volume of 100 µl in TC199 medium (without supplements) by incubating the sample for 10 min at room temperature according to the instructions of the manufacturer. .. After an incubation for 3 h, cells were washed with PBS, 2.5 ml of fresh 20% culture medium was added and cells were then grown for a further 48 h with renewal of the growth medium after 24 h. Luciferase activities were measured in a luminometer, (Biocounter M2010, Lumac BV, The Netherlands) using the Dual-Luciferase Reporter Assay System (Promega) following the instructions of the manufacturer.

Article Title: Mutations Affecting the BHLHA9 DNA-Binding Domain Cause MSSD, Mesoaxial Synostotic Syndactyly with Phalangeal Reduction, Malik-Percin Type
Article Snippet: .. After 24 hr, for each of the wells, transfection complexes composed of 3 ml Attractene (QIAGEN) and 500 ng DNA of recombinant constructs expressing human wild-type or mutant HsBHLHA9_MYC/DDK and/or HsTCF4_GFP fusion proteins were mixed in 120 μl DMEM without serum, incubated for 20 min at room temperature to allow formation of transfection complexes, and subsequently added drop wise to the cells in 2 ml fresh DMEM_10%. .. The cells with the transfection complexes were incubated under normal growth conditions.

Incubation:

Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy
Article Snippet: .. Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days. .. Parallel incubations were carried out by incubating the cells in the transfection reagent alone for 8 hours followed by incubation in 5 mM or 20 mM glucose media. (Our previous studies have shown that control plasmid vectors [pGL3] or scrambled siRNA have no effect on retinal endothelial cells , ).

Article Title: IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling
Article Snippet: .. Wnt5A Silencing To knockdown Wnt5A expression, HCAEC grown up to 80% confluency in 24-well plates were incubated with transfection complexes formed from Wnt5A-siRNA (5 nM; Hs_Wnt5A_6, Cat. No. SI03025596; Qiagen) and 6 μL/mL HiPerFect Transfection reagent (Qiagen GmbH, Hilden, Germany) in EBM-2 basal medium (Clonetics, Lonza, USA) for 24 h. After splitting using Trypsin EDTA, transfected cells were seeded onto collagen coated 6 well plates, retransfected for an additional round as above and incubated until the cells reached 90% confluency. .. HCAEC were also transfected with validated AllStars negative control siRNA (Qiagen) in parallel to control for ‘off target’ effects of siRNAs.

Article Title: Mutations Affecting the BHLHA9 DNA-Binding Domain Cause MSSD, Mesoaxial Synostotic Syndactyly with Phalangeal Reduction, Malik-Percin Type
Article Snippet: .. After 24 hr, for each of the wells, transfection complexes composed of 3 ml Attractene (QIAGEN) and 500 ng DNA of recombinant constructs expressing human wild-type or mutant HsBHLHA9_MYC/DDK and/or HsTCF4_GFP fusion proteins were mixed in 120 μl DMEM without serum, incubated for 20 min at room temperature to allow formation of transfection complexes, and subsequently added drop wise to the cells in 2 ml fresh DMEM_10%. .. The cells with the transfection complexes were incubated under normal growth conditions.

Article Title: Oxidative damage of mitochondrial DNA in diabetes and its protection by manganese superoxide dismutase
Article Snippet: .. Briefly, transfection complexes were formed using Effectene transfection reagent (Qiagen, Valencia, CA) according to the manufacturer’s instructions and incubated for 8 h The cells were rinsed with DMEM and incubated in 5 mM (normal) or 20 mM (high) glucose media. .. Parallel incubations were carried out using the transfection reagent alone (Mock).

Expressing:

Article Title: High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity
Article Snippet: .. Transfection complexes containing 1 µg of promoter construct DNA, 1 µg of HMGA2 expression plasmid, 250 ng of pRL-TK and 10 µl of SuperFect transfection reagent (Qiagen, Hilden, Germany) were formed in a total volume of 100 µl in TC199 medium (without supplements) by incubating the sample for 10 min at room temperature according to the instructions of the manufacturer. .. After an incubation for 3 h, cells were washed with PBS, 2.5 ml of fresh 20% culture medium was added and cells were then grown for a further 48 h with renewal of the growth medium after 24 h. Luciferase activities were measured in a luminometer, (Biocounter M2010, Lumac BV, The Netherlands) using the Dual-Luciferase Reporter Assay System (Promega) following the instructions of the manufacturer.

Article Title: IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling
Article Snippet: .. Wnt5A Silencing To knockdown Wnt5A expression, HCAEC grown up to 80% confluency in 24-well plates were incubated with transfection complexes formed from Wnt5A-siRNA (5 nM; Hs_Wnt5A_6, Cat. No. SI03025596; Qiagen) and 6 μL/mL HiPerFect Transfection reagent (Qiagen GmbH, Hilden, Germany) in EBM-2 basal medium (Clonetics, Lonza, USA) for 24 h. After splitting using Trypsin EDTA, transfected cells were seeded onto collagen coated 6 well plates, retransfected for an additional round as above and incubated until the cells reached 90% confluency. .. HCAEC were also transfected with validated AllStars negative control siRNA (Qiagen) in parallel to control for ‘off target’ effects of siRNAs.

Article Title: Mutations Affecting the BHLHA9 DNA-Binding Domain Cause MSSD, Mesoaxial Synostotic Syndactyly with Phalangeal Reduction, Malik-Percin Type
Article Snippet: .. After 24 hr, for each of the wells, transfection complexes composed of 3 ml Attractene (QIAGEN) and 500 ng DNA of recombinant constructs expressing human wild-type or mutant HsBHLHA9_MYC/DDK and/or HsTCF4_GFP fusion proteins were mixed in 120 μl DMEM without serum, incubated for 20 min at room temperature to allow formation of transfection complexes, and subsequently added drop wise to the cells in 2 ml fresh DMEM_10%. .. The cells with the transfection complexes were incubated under normal growth conditions.

Recombinant:

Article Title: Mutations Affecting the BHLHA9 DNA-Binding Domain Cause MSSD, Mesoaxial Synostotic Syndactyly with Phalangeal Reduction, Malik-Percin Type
Article Snippet: .. After 24 hr, for each of the wells, transfection complexes composed of 3 ml Attractene (QIAGEN) and 500 ng DNA of recombinant constructs expressing human wild-type or mutant HsBHLHA9_MYC/DDK and/or HsTCF4_GFP fusion proteins were mixed in 120 μl DMEM without serum, incubated for 20 min at room temperature to allow formation of transfection complexes, and subsequently added drop wise to the cells in 2 ml fresh DMEM_10%. .. The cells with the transfection complexes were incubated under normal growth conditions.

Plasmid Preparation:

Article Title: High mobility group A2 protein and its derivatives bind a specific region of the promoter of DNA repair gene ERCC1 and modulate its activity
Article Snippet: .. Transfection complexes containing 1 µg of promoter construct DNA, 1 µg of HMGA2 expression plasmid, 250 ng of pRL-TK and 10 µl of SuperFect transfection reagent (Qiagen, Hilden, Germany) were formed in a total volume of 100 µl in TC199 medium (without supplements) by incubating the sample for 10 min at room temperature according to the instructions of the manufacturer. .. After an incubation for 3 h, cells were washed with PBS, 2.5 ml of fresh 20% culture medium was added and cells were then grown for a further 48 h with renewal of the growth medium after 24 h. Luciferase activities were measured in a luminometer, (Biocounter M2010, Lumac BV, The Netherlands) using the Dual-Luciferase Reporter Assay System (Promega) following the instructions of the manufacturer.

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    Qiagen hiperfect
    Gene expression profiles of siRNA treated mice using TLDA. ( a ) cytokines genes, ( b ) chemokine genes and ( c ) cell surface molecules in spleen of mice from 3 groups- (1) CHPV infected control (2) CHPV infected and P-2 siRNA treated and (3) <t>Hiperfect</t> + P-2 siRNA inoculated control. Results are expressed as means ± SD.
    Hiperfect, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Qiagen transfection complexes
    Exosome concentration affects electroporation efficiency. To determine the optimal exosome concentration for <t>transfection,</t> exosomes at four different concentrations (0.25–4 µg/µl) were mixed with an Alexa Fluor 488-tagged siRNA (2 nmol/ml) and electroporated. Samples containing exosomes and siRNA, without an electric pulse, were used as a negative control. To eliminate the excess siRNA, the exosomes were purified using latex beads and the presence of siRNA in the exosomes was determined by FACS analysis.
    Transfection Complexes, supplied by Qiagen, used in various techniques. Bioz Stars score: 92/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Qiagen transient transfection
    Schematic representation and expression of GFP-tagged TSC2 constructs in the TSC2−/− cells. (A) TSC2 includes leucine zipper (LZ), two coiled-coiled (CC), two transcription-activating domains (TAD), GAP homology (GAP), and calmodulin (CaM)-binding domain. (B) To identify expression of TSC2 mutants, cells were transfected with pEGFP vectors expressing GFP-tagged TSC2, N-TSC2, C-TSC2, TSC2-HBD, TSC2-ΔHBD, or control GFP. After 24 h of transient <t>transfection,</t> cells were lysed, and whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-GFP antiserum. TSC2-positive rat TRKE cells, used as a positive control, were lysed and subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-TSC2 antibody.
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    Gene expression profiles of siRNA treated mice using TLDA. ( a ) cytokines genes, ( b ) chemokine genes and ( c ) cell surface molecules in spleen of mice from 3 groups- (1) CHPV infected control (2) CHPV infected and P-2 siRNA treated and (3) Hiperfect + P-2 siRNA inoculated control. Results are expressed as means ± SD.

    Journal: PLoS ONE

    Article Title: Intracranial Administration of P Gene siRNA Protects Mice from Lethal Chandipura Virus Encephalitis

    doi: 10.1371/journal.pone.0008615

    Figure Lengend Snippet: Gene expression profiles of siRNA treated mice using TLDA. ( a ) cytokines genes, ( b ) chemokine genes and ( c ) cell surface molecules in spleen of mice from 3 groups- (1) CHPV infected control (2) CHPV infected and P-2 siRNA treated and (3) Hiperfect + P-2 siRNA inoculated control. Results are expressed as means ± SD.

    Article Snippet: For experiments using siRNA, siRNAs were complexed with HiPerfect™ (QIAGEN, Valencia CA) according to the manufacturer's instructions and were administered at the same site once simultaneously or at different intervals.

    Techniques: Expressing, Mouse Assay, TLDA Assay, Infection

    Exosome concentration affects electroporation efficiency. To determine the optimal exosome concentration for transfection, exosomes at four different concentrations (0.25–4 µg/µl) were mixed with an Alexa Fluor 488-tagged siRNA (2 nmol/ml) and electroporated. Samples containing exosomes and siRNA, without an electric pulse, were used as a negative control. To eliminate the excess siRNA, the exosomes were purified using latex beads and the presence of siRNA in the exosomes was determined by FACS analysis.

    Journal: Nucleic Acids Research

    Article Title: Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

    doi: 10.1093/nar/gks463

    Figure Lengend Snippet: Exosome concentration affects electroporation efficiency. To determine the optimal exosome concentration for transfection, exosomes at four different concentrations (0.25–4 µg/µl) were mixed with an Alexa Fluor 488-tagged siRNA (2 nmol/ml) and electroporated. Samples containing exosomes and siRNA, without an electric pulse, were used as a negative control. To eliminate the excess siRNA, the exosomes were purified using latex beads and the presence of siRNA in the exosomes was determined by FACS analysis.

    Article Snippet: To examine the efficiency of chemical transfection, a siRNA against MAPK1 was mixed with transfection reagent to allow for the formation of transfection complexes (siRNA embedded in lipid micelles), according to the manufacturer’s instructions (RNAi Human/Mouse Starter Kit, Quiagen).

    Techniques: Concentration Assay, Electroporation, Transfection, Negative Control, Purification, FACS

    Flow cytometry and northern slot blot of exosomes following chemical transfection. siRNA was embedded into lipid micelles according to the manufacturer’s instructions. The embedded siRNA was mixed with exosomes and incubated overnight. To eliminate the excess siRNA embedded in transfection complexes, the exosomes were purified using latex beads. The beads were washed twice with PBS to eliminate the excess of un-specific bound siRNA molecules. The presence of fluorescently labeled siRNA was determined by flow cytometry and northern blotting (slot blot). Panel A–D shows the results of flow cytometry for the detection of Alexa Fluor-488 labeled siRNA, beads only ( A ), beads plus siRNA ( B ), beads plus siRNA embedded in lipid micelles (negative control; C ) and chemically transfected exosomes ( D ). Panels E and F show the results of northern blotting using a digoxigenin-labeled probe against the administered MAPK1 siRNA. Panel E shows the presence of siRNA in exosomes and micelles. The chemically transfected exosomes were mixed with their parental cells HTB-177 and the delivery of siRNA from the exosomes to cells was determined and compared with direct transfection of cells ( F ). Non-transfected exosomes and cells were used as negative control.

    Journal: Nucleic Acids Research

    Article Title: Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

    doi: 10.1093/nar/gks463

    Figure Lengend Snippet: Flow cytometry and northern slot blot of exosomes following chemical transfection. siRNA was embedded into lipid micelles according to the manufacturer’s instructions. The embedded siRNA was mixed with exosomes and incubated overnight. To eliminate the excess siRNA embedded in transfection complexes, the exosomes were purified using latex beads. The beads were washed twice with PBS to eliminate the excess of un-specific bound siRNA molecules. The presence of fluorescently labeled siRNA was determined by flow cytometry and northern blotting (slot blot). Panel A–D shows the results of flow cytometry for the detection of Alexa Fluor-488 labeled siRNA, beads only ( A ), beads plus siRNA ( B ), beads plus siRNA embedded in lipid micelles (negative control; C ) and chemically transfected exosomes ( D ). Panels E and F show the results of northern blotting using a digoxigenin-labeled probe against the administered MAPK1 siRNA. Panel E shows the presence of siRNA in exosomes and micelles. The chemically transfected exosomes were mixed with their parental cells HTB-177 and the delivery of siRNA from the exosomes to cells was determined and compared with direct transfection of cells ( F ). Non-transfected exosomes and cells were used as negative control.

    Article Snippet: To examine the efficiency of chemical transfection, a siRNA against MAPK1 was mixed with transfection reagent to allow for the formation of transfection complexes (siRNA embedded in lipid micelles), according to the manufacturer’s instructions (RNAi Human/Mouse Starter Kit, Quiagen).

    Techniques: Flow Cytometry, Cytometry, Northern Blot, Dot Blot, Transfection, Incubation, Purification, Labeling, Negative Control

    GAPDH in retinal endothelial cells. Retinal endothelial cells were transfected using 0.5 to 5.0 μg GAPDH expression plasmid for 8 hours. ( a ) GAPDH activity was measured spectrophotometrically to determine the degree of transfection. ( b , c ) BRECs were transfected with 2.5 μg GAPDH plasmid or no plasmid but with transfection reagent alone (R) for 8 hours and were incubated for 4 days in 5 mM or 20 mM glucose media. ( b ) GAPDH abundance was assessed by Western blot analysis, and ( c ) its glycolytic activity was assessed by measuring the production of NADH at 340 nm. Each parameter was measured in duplicate using four different cell preparations. Results are presented as mean ± SD of the values obtained from three different cell preparations, with each measurement made in duplicate. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected BRECs incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone, followed by incubation in 5 mM or 20 mM glucose, respectively. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy

    doi: 10.1167/iovs.09-4171

    Figure Lengend Snippet: GAPDH in retinal endothelial cells. Retinal endothelial cells were transfected using 0.5 to 5.0 μg GAPDH expression plasmid for 8 hours. ( a ) GAPDH activity was measured spectrophotometrically to determine the degree of transfection. ( b , c ) BRECs were transfected with 2.5 μg GAPDH plasmid or no plasmid but with transfection reagent alone (R) for 8 hours and were incubated for 4 days in 5 mM or 20 mM glucose media. ( b ) GAPDH abundance was assessed by Western blot analysis, and ( c ) its glycolytic activity was assessed by measuring the production of NADH at 340 nm. Each parameter was measured in duplicate using four different cell preparations. Results are presented as mean ± SD of the values obtained from three different cell preparations, with each measurement made in duplicate. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected BRECs incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone, followed by incubation in 5 mM or 20 mM glucose, respectively. * P

    Article Snippet: Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days.

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

    Effect of GAPDH overexpression on endothelial cell death: GAPDH-transfected BRECs (using 2.5 μg plasmid) or untransfected BRECs were incubated in 5 mM or 20 mM glucose for 4 days. ( a ) Apoptosis was determined by cytoplasmic histone-associated DNA fragments using ELISA. The graph shows the mean ± SD adjusted to the total DNA in each sample. ( b ) Activation of apoptosis execution enzyme caspase-3 was determined in the cells by measuring the cleavage of the substrate Ac-DEVD-pNA. Each experiment was repeated with at least three different BREC preparations, and measurements were made in duplicate. ( c ) The appearance of a 85-kDa band of PARP was determined by Western blot technique, and β-actin was used as a loading standard. Western blot is representative of four different experiments. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 20+Fe, 20 mM glucose and 2.5 μM FeTPPS; 20+PJ, 20 mM glucose and 1.0 μM PJ34; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone before incubation with 5 mM or 20 mM glucose, respectively. The values, represented as mean ± SD, obtained with 5 mM glucose were considered 100%. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy

    doi: 10.1167/iovs.09-4171

    Figure Lengend Snippet: Effect of GAPDH overexpression on endothelial cell death: GAPDH-transfected BRECs (using 2.5 μg plasmid) or untransfected BRECs were incubated in 5 mM or 20 mM glucose for 4 days. ( a ) Apoptosis was determined by cytoplasmic histone-associated DNA fragments using ELISA. The graph shows the mean ± SD adjusted to the total DNA in each sample. ( b ) Activation of apoptosis execution enzyme caspase-3 was determined in the cells by measuring the cleavage of the substrate Ac-DEVD-pNA. Each experiment was repeated with at least three different BREC preparations, and measurements were made in duplicate. ( c ) The appearance of a 85-kDa band of PARP was determined by Western blot technique, and β-actin was used as a loading standard. Western blot is representative of four different experiments. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 20+Fe, 20 mM glucose and 2.5 μM FeTPPS; 20+PJ, 20 mM glucose and 1.0 μM PJ34; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 5+R and 20+R, BRECs treated with the transfection reagent alone before incubation with 5 mM or 20 mM glucose, respectively. The values, represented as mean ± SD, obtained with 5 mM glucose were considered 100%. * P

    Article Snippet: Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Incubation, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot

    Subcellular distribution of GAPDH. BRECs either transfected with 2.5 μg GAPDH plasmid or left untransfected were incubated for 4 days in 5 mM or 20 mM glucose media. The nuclear fraction was prepared by centrifugation, and the expression of GAPDH was determined by Western blot analysis using histone 2B as a loading control. Western blot represents results obtained from three or four different experiments, with each measurement made in duplicate. Histogram represents mean ± SD. GAPDH band intensity adjusted to H2B band intensity, and the values obtained from BRECs incubated in 5 mM glucose are considered 100%. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 20+R, BRECs treated with the transfection reagent alone before incubation with 20 mM glucose. * P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Glyceraldehyde-3-Phosphate Dehydrogenase in Retinal Microvasculature: Implications for the Development and Progression of Diabetic Retinopathy

    doi: 10.1167/iovs.09-4171

    Figure Lengend Snippet: Subcellular distribution of GAPDH. BRECs either transfected with 2.5 μg GAPDH plasmid or left untransfected were incubated for 4 days in 5 mM or 20 mM glucose media. The nuclear fraction was prepared by centrifugation, and the expression of GAPDH was determined by Western blot analysis using histone 2B as a loading control. Western blot represents results obtained from three or four different experiments, with each measurement made in duplicate. Histogram represents mean ± SD. GAPDH band intensity adjusted to H2B band intensity, and the values obtained from BRECs incubated in 5 mM glucose are considered 100%. 5 mM, 5 mM glucose; 20 mM, 20 mM glucose; 5+GAP and 20+GAP, GAPDH-transfected cells incubated in 5 mM or 20 mM glucose, respectively; 20+R, BRECs treated with the transfection reagent alone before incubation with 20 mM glucose. * P

    Article Snippet: Transfection complexes were formed with transfection reagent (Effectene; Qiagen, Valencia, CA) and were incubated with BRECs for 8 hours before incubation in 5 mM or 20 mM glucose media for 4 days.

    Techniques: Transfection, Plasmid Preparation, Incubation, Centrifugation, Expressing, Western Blot

    Schematic representation and expression of GFP-tagged TSC2 constructs in the TSC2−/− cells. (A) TSC2 includes leucine zipper (LZ), two coiled-coiled (CC), two transcription-activating domains (TAD), GAP homology (GAP), and calmodulin (CaM)-binding domain. (B) To identify expression of TSC2 mutants, cells were transfected with pEGFP vectors expressing GFP-tagged TSC2, N-TSC2, C-TSC2, TSC2-HBD, TSC2-ΔHBD, or control GFP. After 24 h of transient transfection, cells were lysed, and whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-GFP antiserum. TSC2-positive rat TRKE cells, used as a positive control, were lysed and subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-TSC2 antibody.

    Journal: The Journal of Cell Biology

    Article Title: TSC2 modulates actin cytoskeleton and focal adhesion through TSC1-binding domain and the Rac1 GTPase

    doi: 10.1083/jcb.200405130

    Figure Lengend Snippet: Schematic representation and expression of GFP-tagged TSC2 constructs in the TSC2−/− cells. (A) TSC2 includes leucine zipper (LZ), two coiled-coiled (CC), two transcription-activating domains (TAD), GAP homology (GAP), and calmodulin (CaM)-binding domain. (B) To identify expression of TSC2 mutants, cells were transfected with pEGFP vectors expressing GFP-tagged TSC2, N-TSC2, C-TSC2, TSC2-HBD, TSC2-ΔHBD, or control GFP. After 24 h of transient transfection, cells were lysed, and whole cell lysates were subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-GFP antiserum. TSC2-positive rat TRKE cells, used as a positive control, were lysed and subjected to 8% SDS-PAGE followed by immunoblot analysis with anti-TSC2 antibody.

    Article Snippet: Transient transfection and replication-deficient adenovirus infection Transient transfection was performed using the Effectene transfection reagent (QIAGEN) according to the manufacturer's protocol.

    Techniques: Expressing, Construct, Chick Chorioallantoic Membrane Assay, Binding Assay, Transfection, SDS Page, Positive Control