Structured Review

Promega pcdna
Fig. 2. ICSBP down-regulates PU.1 induced transactivation of the Dab2 promoter. ( A ) K562 cells were transfected with pGL3-Basic containing a 2 kb fragment of the mouse Dab2 promoter together with <t>pcDNA</t> or pcDNA-ICSBP and the reference vector <t>pRL-TK.</t> Luciferase reporter gene activities were measured as described in Materials and methods. ( B ) CV-1 cells were transiently transfected with a Dab2 promoter driven luciferase reporter gene (300 ng) along with the following expression vectors: PU.1 200 ng; ICSBP 200 ng or 600 ng. Empty vector DNA was added so that each reaction contained a total of 800 ng of expression plasmid. ( C ) Endogenously expressed PU.1 binds to the Dab2 regulatory sequence. K562 nuclear extracts were incubated with the immobilized Dab2 promoter DNA, and the bound fraction was analyzed by western blotting using antibodies as indicated. ( D ) ICSBP and PU.1 bind to the Dab2 promoter. Magnetic beads containing the 2 kb mouse Dab2 promoter were incubated with nuclear extracts of CV-1 cells, which were supplemented with 10 µl of 35 S-labeled ICSBP, PU.1, or both ICSBP and PU.1. The beads were washed and the eluates were subjected to SDS–PAGE and autoradiography. 35 S-labeled protein (1 µl) was used as an input control. Probing of the membrane with an anti-Stat3 antibody confirmed the specificity of the employed assay.
Pcdna, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Disabled-2 is transcriptionally regulated by ICSBP and augments macrophage spreading and adhesion"

Article Title: Disabled-2 is transcriptionally regulated by ICSBP and augments macrophage spreading and adhesion

Journal: The EMBO Journal

doi: 10.1093/emboj/21.3.211

Fig. 2. ICSBP down-regulates PU.1 induced transactivation of the Dab2 promoter. ( A ) K562 cells were transfected with pGL3-Basic containing a 2 kb fragment of the mouse Dab2 promoter together with pcDNA or pcDNA-ICSBP and the reference vector pRL-TK. Luciferase reporter gene activities were measured as described in Materials and methods. ( B ) CV-1 cells were transiently transfected with a Dab2 promoter driven luciferase reporter gene (300 ng) along with the following expression vectors: PU.1 200 ng; ICSBP 200 ng or 600 ng. Empty vector DNA was added so that each reaction contained a total of 800 ng of expression plasmid. ( C ) Endogenously expressed PU.1 binds to the Dab2 regulatory sequence. K562 nuclear extracts were incubated with the immobilized Dab2 promoter DNA, and the bound fraction was analyzed by western blotting using antibodies as indicated. ( D ) ICSBP and PU.1 bind to the Dab2 promoter. Magnetic beads containing the 2 kb mouse Dab2 promoter were incubated with nuclear extracts of CV-1 cells, which were supplemented with 10 µl of 35 S-labeled ICSBP, PU.1, or both ICSBP and PU.1. The beads were washed and the eluates were subjected to SDS–PAGE and autoradiography. 35 S-labeled protein (1 µl) was used as an input control. Probing of the membrane with an anti-Stat3 antibody confirmed the specificity of the employed assay.
Figure Legend Snippet: Fig. 2. ICSBP down-regulates PU.1 induced transactivation of the Dab2 promoter. ( A ) K562 cells were transfected with pGL3-Basic containing a 2 kb fragment of the mouse Dab2 promoter together with pcDNA or pcDNA-ICSBP and the reference vector pRL-TK. Luciferase reporter gene activities were measured as described in Materials and methods. ( B ) CV-1 cells were transiently transfected with a Dab2 promoter driven luciferase reporter gene (300 ng) along with the following expression vectors: PU.1 200 ng; ICSBP 200 ng or 600 ng. Empty vector DNA was added so that each reaction contained a total of 800 ng of expression plasmid. ( C ) Endogenously expressed PU.1 binds to the Dab2 regulatory sequence. K562 nuclear extracts were incubated with the immobilized Dab2 promoter DNA, and the bound fraction was analyzed by western blotting using antibodies as indicated. ( D ) ICSBP and PU.1 bind to the Dab2 promoter. Magnetic beads containing the 2 kb mouse Dab2 promoter were incubated with nuclear extracts of CV-1 cells, which were supplemented with 10 µl of 35 S-labeled ICSBP, PU.1, or both ICSBP and PU.1. The beads were washed and the eluates were subjected to SDS–PAGE and autoradiography. 35 S-labeled protein (1 µl) was used as an input control. Probing of the membrane with an anti-Stat3 antibody confirmed the specificity of the employed assay.

Techniques Used: Transfection, Plasmid Preparation, Luciferase, Expressing, Sequencing, Incubation, Western Blot, Magnetic Beads, Labeling, SDS Page, Autoradiography

2) Product Images from "HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN"

Article Title: HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN

Journal: Scientific Reports

doi: 10.1038/srep40089

HBx inhibits cell apoptosis through up-regulates miR-181a and in turn down-regulates PTEN. Cell apoptosis was analyzed by using the Annexin V-PE apoptosis detection kit. Apoptosis rate of HepG2 cells transfected with miR-181a and the corresponding controls, versus the blank group ( a Blank, b pRNAT, c miR-181a). Apoptosis rate of HepG2 cells transfected with pHBx and the corresponding control ( d pcDNA3.0, e HBx), cotransfected with pHBx and miR-181a inhibitor and the corresponding control ( f HBx + NC, g HBx + miR-181In). Apoptosis rate of HepG2 cells transfected with PTEN, pHBx cotransfected with PTEN, pcDNA3.0 cotransfected with PTEN ( h PTEN, i pcDNA + PTEN, j . HBx + PTEN). k Histogram of the apoptosis rate from group a to j . Apoptosis rate of HepG2.2.15 cells transfected with miR-181a inhibitor (miR-181In) and the corresponding controls (NC), versus the blank group ( A Blank, B NC, C miR-181In). ( D ) Histogram of the apoptosis rate from group A to C . (n = 3, * p
Figure Legend Snippet: HBx inhibits cell apoptosis through up-regulates miR-181a and in turn down-regulates PTEN. Cell apoptosis was analyzed by using the Annexin V-PE apoptosis detection kit. Apoptosis rate of HepG2 cells transfected with miR-181a and the corresponding controls, versus the blank group ( a Blank, b pRNAT, c miR-181a). Apoptosis rate of HepG2 cells transfected with pHBx and the corresponding control ( d pcDNA3.0, e HBx), cotransfected with pHBx and miR-181a inhibitor and the corresponding control ( f HBx + NC, g HBx + miR-181In). Apoptosis rate of HepG2 cells transfected with PTEN, pHBx cotransfected with PTEN, pcDNA3.0 cotransfected with PTEN ( h PTEN, i pcDNA + PTEN, j . HBx + PTEN). k Histogram of the apoptosis rate from group a to j . Apoptosis rate of HepG2.2.15 cells transfected with miR-181a inhibitor (miR-181In) and the corresponding controls (NC), versus the blank group ( A Blank, B NC, C miR-181In). ( D ) Histogram of the apoptosis rate from group A to C . (n = 3, * p

Techniques Used: Transfection

3) Product Images from "Sox9 Transcriptionally Represses Spp1 to Prevent Matrix Mineralization in Maturing Heart Valves and Chondrocytes"

Article Title: Sox9 Transcriptionally Represses Spp1 to Prevent Matrix Mineralization in Maturing Heart Valves and Chondrocytes

Journal: PLoS ONE

doi: 10.1371/journal.pone.0026769

Luciferase assays of Sox9 function on Spp1 activity. HEK293 (A) and MC-3T3 (B) cells were co-tranfected with 200 ng of the luciferase reporter construct pGL3 containing the Spp1 promoter region (no SRY binding sites) (pGL3- spp1 p), the 1599 bp 5’ Spp1 enhancer region containing SRY#1 (pGL3- spp1 En), or both the enhancer and promoter regions (pGL3- spp1 ), along with 200 ng of empty pcDNA (grey bars) or pcDNA containing full length murine Sox9 (pcDNA-Sox9, black bars). In addition, cells were co-transfected with 20 ng of pGL4 for normalization. Luciferase activity of pGL3- spp1 with empty pcDNA was set to 100%. Note repression of pGL3- spp1 activity (63%±8% (A) and 60.3%±12.6% (B) in the presence of pcDNA-Sox9. Cells were also co-transfected with pcDNA or pcDNA-Sox9 in the presence of pGL3- spp1 in which the SRY#1 had been mutated from TA CAAA G to TA GCAG G (pGL3- spp1 mut). Note that pcDNA-Sox9 could not significantly repress pGL3- spp1 mut unlike pGL3- spp1 . *Student's t−test
Figure Legend Snippet: Luciferase assays of Sox9 function on Spp1 activity. HEK293 (A) and MC-3T3 (B) cells were co-tranfected with 200 ng of the luciferase reporter construct pGL3 containing the Spp1 promoter region (no SRY binding sites) (pGL3- spp1 p), the 1599 bp 5’ Spp1 enhancer region containing SRY#1 (pGL3- spp1 En), or both the enhancer and promoter regions (pGL3- spp1 ), along with 200 ng of empty pcDNA (grey bars) or pcDNA containing full length murine Sox9 (pcDNA-Sox9, black bars). In addition, cells were co-transfected with 20 ng of pGL4 for normalization. Luciferase activity of pGL3- spp1 with empty pcDNA was set to 100%. Note repression of pGL3- spp1 activity (63%±8% (A) and 60.3%±12.6% (B) in the presence of pcDNA-Sox9. Cells were also co-transfected with pcDNA or pcDNA-Sox9 in the presence of pGL3- spp1 in which the SRY#1 had been mutated from TA CAAA G to TA GCAG G (pGL3- spp1 mut). Note that pcDNA-Sox9 could not significantly repress pGL3- spp1 mut unlike pGL3- spp1 . *Student's t−test

Techniques Used: Luciferase, Activity Assay, Construct, Binding Assay, Transfection

4) Product Images from "Interaction between hnRNPA1 and I?B? Is Required for Maximal Activation of NF-?B-Dependent Transcription"

Article Title: Interaction between hnRNPA1 and I?B? Is Required for Maximal Activation of NF-?B-Dependent Transcription

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.10.3482-3490.2001

hnRNPA1 enhances IκBα processing in response to LMP-1 activation. (A, B, and C) IκBα wild type (WT) and the S32A S36A, 1–292, and 1–303 constructs were electroporated with pcDNA 3 empty vector or pcDNA 3 expression constructs containing either hnRNPA1 cDNA or the indicated truncation mutants into CB3 cells which do not express hnRNPA1. To provide an NF-κB activation signal, cells were electroporated with an expression construct containing the cDNA for EBV LMP-1 or empty vector. To control for the level of transfection between the different conditions, the cells were transfected with an expression construct containing the cDNA for pyruvate kinase with a myc tag. Sixteen hours posttransfection the cells were lysed and separated by SDS–10% PAGE. Following separation, cells were transferred to a polyvinylidene difluoride membrane and analyzed using the SV5 monoclonal antibody to SV5-tagged proteins and the myc monoclonal antibody to myc-tagged proteins.
Figure Legend Snippet: hnRNPA1 enhances IκBα processing in response to LMP-1 activation. (A, B, and C) IκBα wild type (WT) and the S32A S36A, 1–292, and 1–303 constructs were electroporated with pcDNA 3 empty vector or pcDNA 3 expression constructs containing either hnRNPA1 cDNA or the indicated truncation mutants into CB3 cells which do not express hnRNPA1. To provide an NF-κB activation signal, cells were electroporated with an expression construct containing the cDNA for EBV LMP-1 or empty vector. To control for the level of transfection between the different conditions, the cells were transfected with an expression construct containing the cDNA for pyruvate kinase with a myc tag. Sixteen hours posttransfection the cells were lysed and separated by SDS–10% PAGE. Following separation, cells were transferred to a polyvinylidene difluoride membrane and analyzed using the SV5 monoclonal antibody to SV5-tagged proteins and the myc monoclonal antibody to myc-tagged proteins.

Techniques Used: Activation Assay, Construct, Plasmid Preparation, Expressing, Transfection, Polyacrylamide Gel Electrophoresis

Related Articles

Clone Assay:

Article Title: Human Parainfluenza Virus Type 3 Infections in Patients with Hematopoietic Stem Cell Transplants: the Mode of Nosocomial Infections and Prognosis.
Article Snippet: .. They were, subsequently, sequenced through cloning into pcDNA (Promega, Madison, WI) using the universal primers for the vector, and the virus type was determined. .. Determination of the partial hemagglutinin-neuraminidase (HN) gene of PIV3: The viral RNA was extracted from the PIV3 stock isolated using a High Pure Viral RNA Kit (Roche), and a reverse-transcription PCR was conducted using an Illustra Ready-To-Go RT-PCR Beads kit (GE, Fairfield, CT, USA) with primers targeting part of the HN gene of PIV3 (30), which was used in previous epidemiological studies of PIV3 outbreaks (14).

Transfection:

Article Title: Mapping ER? Genomic Binding Sites Reveals Unique Genomic Features and Identifies EBF1 as an ER? Interactor
Article Snippet: .. Cell Proliferation Assay C4–12/Flag.ERβ cells were transfected in 24-well plates with pcDNA or EBF1 plasmids using FuGENE (Promega). .. Cell confluency, used to quantify cell growth, was measured in an Incucyte FLR live content imaging system (Essen Bioscience).

Luciferase:

Article Title: HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN
Article Snippet: .. Dual-luciferase assay To study the effects of HBx and miR-181a on the activity of 3′untranslated regions (3′-UTRs) of PTEN which contains an intact miR-181a recognition sequence, the expression plasmids for HBx or pcDNA, 3′untranslated regions of PTEN-luciferase reporter plasmids (pGL-PTEN) and Renilla luciferase (RL, Promega) expression vector (PRL-TK) were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates; the incremental dose of miR-181a or pRNAT vector (1,2,4 μg, respectively) was co-transfected with pGL-PTEN (0.5 μg) and PRL-TK(0.1 μg) into HepG2 cells. .. To investigate the effects of HBx on the activity of the promoter of miR-181a, the expression plasmids for HBx or pcDNA, pGL181a-P and PRL-TK were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates.

Article Title: Sox9 Transcriptionally Represses Spp1 to Prevent Matrix Mineralization in Maturing Heart Valves and Chondrocytes
Article Snippet: .. 200 ng of pGL3-spp1, pGL3-spp1 mut or pGL3 (200 ng/well), and pcDNA or pcDNA-Sox9 (200 ng/well), were co-transfected into each well, along with 20 ng of pGL4 (Renilla luciferase, Promega). .. All transfections were performed in 0.5 mL OptiMem for 4 hours before the addition of 0.5 mL DMEM (Sigma) supplemented with 4 mM L-Glutamine and 10% FBS.

In Vitro:

Article Title: Liaisons between Survivin and Plk1 during Cell Division and Cell Death *
Article Snippet: .. For GST pull-down experiments, the GST-tagged proteins bound to beads were incubated with an in vitro translated “partner” protein expressed from pcDNA or pBluescript using a TNT-T7 transcription-translation kit (Promega) and [35 S]methionine (PerkinElmer Life Sciences) as tracer (see Ref. for detailed methods). .. Fluorescence Microscopy To localize Plk1 kinase, cells were fixed with 4% formaldehyde in PBS for 5 min, washed with PBS, permeabilized with 0.15% Triton for 2 min, and blocked with 1% bovine serum albumin in PBS for 15 min and immunoprobed with anti-Plk1 (AbCam, Ab14209, 1:100), anti-CENPC (AbCam, Ab50974, 1:250), anti-Aurora-B (AbCam, Ab2254, 1:1000), anti-borealin (polyclonal, in house), or anti-BubR1 (1:250, gift from S. S. Taylor (Manchester, UK)) antibodies, followed by Texas Red anti-rabbit, anti-sheep, or anti-mouse antibodies as appropriate (1:200, Vector Laboratories).

Incubation:

Article Title: Liaisons between Survivin and Plk1 during Cell Division and Cell Death *
Article Snippet: .. For GST pull-down experiments, the GST-tagged proteins bound to beads were incubated with an in vitro translated “partner” protein expressed from pcDNA or pBluescript using a TNT-T7 transcription-translation kit (Promega) and [35 S]methionine (PerkinElmer Life Sciences) as tracer (see Ref. for detailed methods). .. Fluorescence Microscopy To localize Plk1 kinase, cells were fixed with 4% formaldehyde in PBS for 5 min, washed with PBS, permeabilized with 0.15% Triton for 2 min, and blocked with 1% bovine serum albumin in PBS for 15 min and immunoprobed with anti-Plk1 (AbCam, Ab14209, 1:100), anti-CENPC (AbCam, Ab50974, 1:250), anti-Aurora-B (AbCam, Ab2254, 1:1000), anti-borealin (polyclonal, in house), or anti-BubR1 (1:250, gift from S. S. Taylor (Manchester, UK)) antibodies, followed by Texas Red anti-rabbit, anti-sheep, or anti-mouse antibodies as appropriate (1:200, Vector Laboratories).

Cell Culture:

Article Title: HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN
Article Snippet: .. Dual-luciferase assay To study the effects of HBx and miR-181a on the activity of 3′untranslated regions (3′-UTRs) of PTEN which contains an intact miR-181a recognition sequence, the expression plasmids for HBx or pcDNA, 3′untranslated regions of PTEN-luciferase reporter plasmids (pGL-PTEN) and Renilla luciferase (RL, Promega) expression vector (PRL-TK) were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates; the incremental dose of miR-181a or pRNAT vector (1,2,4 μg, respectively) was co-transfected with pGL-PTEN (0.5 μg) and PRL-TK(0.1 μg) into HepG2 cells. .. To investigate the effects of HBx on the activity of the promoter of miR-181a, the expression plasmids for HBx or pcDNA, pGL181a-P and PRL-TK were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates.

Purification:

Article Title: Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night
Article Snippet: .. Plasmids Plasmids CMV5′UTR L1Neo, ΔCMVL1Neo, and untagged CMV5′UTR L1 are from , untagged CMV5′UTR L1 ORF1stop is from , CMVΔ5′UTR L1Blast is from , codon optimized human L1 (co hL1) is from ( ) codon-optimized human ORF1 expression plasmid (ORF1) is from , AluNeo is from ( , ), pCDNA, CMV Fluc, (Promega), 5′UTR Fluc are from , MT1 is from , Untagged ΔCMV L1 was generated by NotI and SalI digest of JM101 L1.3, followed by gel extraction and purification of the L1-containing fragment. .. The purified L1-containing sequence was cloned into pBluescriptII (SK) digested with NotI and SalI.

Plasmid Preparation:

Article Title: Human Parainfluenza Virus Type 3 Infections in Patients with Hematopoietic Stem Cell Transplants: the Mode of Nosocomial Infections and Prognosis.
Article Snippet: .. They were, subsequently, sequenced through cloning into pcDNA (Promega, Madison, WI) using the universal primers for the vector, and the virus type was determined. .. Determination of the partial hemagglutinin-neuraminidase (HN) gene of PIV3: The viral RNA was extracted from the PIV3 stock isolated using a High Pure Viral RNA Kit (Roche), and a reverse-transcription PCR was conducted using an Illustra Ready-To-Go RT-PCR Beads kit (GE, Fairfield, CT, USA) with primers targeting part of the HN gene of PIV3 (30), which was used in previous epidemiological studies of PIV3 outbreaks (14).

Article Title: HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN
Article Snippet: .. Dual-luciferase assay To study the effects of HBx and miR-181a on the activity of 3′untranslated regions (3′-UTRs) of PTEN which contains an intact miR-181a recognition sequence, the expression plasmids for HBx or pcDNA, 3′untranslated regions of PTEN-luciferase reporter plasmids (pGL-PTEN) and Renilla luciferase (RL, Promega) expression vector (PRL-TK) were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates; the incremental dose of miR-181a or pRNAT vector (1,2,4 μg, respectively) was co-transfected with pGL-PTEN (0.5 μg) and PRL-TK(0.1 μg) into HepG2 cells. .. To investigate the effects of HBx on the activity of the promoter of miR-181a, the expression plasmids for HBx or pcDNA, pGL181a-P and PRL-TK were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates.

Article Title: Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night
Article Snippet: .. Plasmids Plasmids CMV5′UTR L1Neo, ΔCMVL1Neo, and untagged CMV5′UTR L1 are from , untagged CMV5′UTR L1 ORF1stop is from , CMVΔ5′UTR L1Blast is from , codon optimized human L1 (co hL1) is from ( ) codon-optimized human ORF1 expression plasmid (ORF1) is from , AluNeo is from ( , ), pCDNA, CMV Fluc, (Promega), 5′UTR Fluc are from , MT1 is from , Untagged ΔCMV L1 was generated by NotI and SalI digest of JM101 L1.3, followed by gel extraction and purification of the L1-containing fragment. .. The purified L1-containing sequence was cloned into pBluescriptII (SK) digested with NotI and SalI.

Generated:

Article Title: Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night
Article Snippet: .. Plasmids Plasmids CMV5′UTR L1Neo, ΔCMVL1Neo, and untagged CMV5′UTR L1 are from , untagged CMV5′UTR L1 ORF1stop is from , CMVΔ5′UTR L1Blast is from , codon optimized human L1 (co hL1) is from ( ) codon-optimized human ORF1 expression plasmid (ORF1) is from , AluNeo is from ( , ), pCDNA, CMV Fluc, (Promega), 5′UTR Fluc are from , MT1 is from , Untagged ΔCMV L1 was generated by NotI and SalI digest of JM101 L1.3, followed by gel extraction and purification of the L1-containing fragment. .. The purified L1-containing sequence was cloned into pBluescriptII (SK) digested with NotI and SalI.

Activity Assay:

Article Title: HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN
Article Snippet: .. Dual-luciferase assay To study the effects of HBx and miR-181a on the activity of 3′untranslated regions (3′-UTRs) of PTEN which contains an intact miR-181a recognition sequence, the expression plasmids for HBx or pcDNA, 3′untranslated regions of PTEN-luciferase reporter plasmids (pGL-PTEN) and Renilla luciferase (RL, Promega) expression vector (PRL-TK) were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates; the incremental dose of miR-181a or pRNAT vector (1,2,4 μg, respectively) was co-transfected with pGL-PTEN (0.5 μg) and PRL-TK(0.1 μg) into HepG2 cells. .. To investigate the effects of HBx on the activity of the promoter of miR-181a, the expression plasmids for HBx or pcDNA, pGL181a-P and PRL-TK were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates.

Construct:

Article Title: Interaction between hnRNPA1 and I?B? Is Required for Maximal Activation of NF-?B-Dependent Transcription
Article Snippet: .. To generate 35 S-labeled IκBα and hnRNPA1 proteins, pCDNA and the appropriate linearized cDNA constructs were used as the template in the TNT coupled-wheat germ extract system (Promega). .. Proteins were translated in a final volume of 50 μl in the presence of 20 μCi of 35 S-labeled methionine (Amersham).

Expressing:

Article Title: HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN
Article Snippet: .. Dual-luciferase assay To study the effects of HBx and miR-181a on the activity of 3′untranslated regions (3′-UTRs) of PTEN which contains an intact miR-181a recognition sequence, the expression plasmids for HBx or pcDNA, 3′untranslated regions of PTEN-luciferase reporter plasmids (pGL-PTEN) and Renilla luciferase (RL, Promega) expression vector (PRL-TK) were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates; the incremental dose of miR-181a or pRNAT vector (1,2,4 μg, respectively) was co-transfected with pGL-PTEN (0.5 μg) and PRL-TK(0.1 μg) into HepG2 cells. .. To investigate the effects of HBx on the activity of the promoter of miR-181a, the expression plasmids for HBx or pcDNA, pGL181a-P and PRL-TK were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates.

Article Title: Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night
Article Snippet: .. Plasmids Plasmids CMV5′UTR L1Neo, ΔCMVL1Neo, and untagged CMV5′UTR L1 are from , untagged CMV5′UTR L1 ORF1stop is from , CMVΔ5′UTR L1Blast is from , codon optimized human L1 (co hL1) is from ( ) codon-optimized human ORF1 expression plasmid (ORF1) is from , AluNeo is from ( , ), pCDNA, CMV Fluc, (Promega), 5′UTR Fluc are from , MT1 is from , Untagged ΔCMV L1 was generated by NotI and SalI digest of JM101 L1.3, followed by gel extraction and purification of the L1-containing fragment. .. The purified L1-containing sequence was cloned into pBluescriptII (SK) digested with NotI and SalI.

Sequencing:

Article Title: HBx promotes cell proliferation by disturbing the cross-talk between miR-181a and PTEN
Article Snippet: .. Dual-luciferase assay To study the effects of HBx and miR-181a on the activity of 3′untranslated regions (3′-UTRs) of PTEN which contains an intact miR-181a recognition sequence, the expression plasmids for HBx or pcDNA, 3′untranslated regions of PTEN-luciferase reporter plasmids (pGL-PTEN) and Renilla luciferase (RL, Promega) expression vector (PRL-TK) were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates; the incremental dose of miR-181a or pRNAT vector (1,2,4 μg, respectively) was co-transfected with pGL-PTEN (0.5 μg) and PRL-TK(0.1 μg) into HepG2 cells. .. To investigate the effects of HBx on the activity of the promoter of miR-181a, the expression plasmids for HBx or pcDNA, pGL181a-P and PRL-TK were mixed (4:0.5:0.1) and co-transfected into HepG2 cells cultured in 6-well plates.

Gel Extraction:

Article Title: Regulation of L1 expression and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light exposure at night
Article Snippet: .. Plasmids Plasmids CMV5′UTR L1Neo, ΔCMVL1Neo, and untagged CMV5′UTR L1 are from , untagged CMV5′UTR L1 ORF1stop is from , CMVΔ5′UTR L1Blast is from , codon optimized human L1 (co hL1) is from ( ) codon-optimized human ORF1 expression plasmid (ORF1) is from , AluNeo is from ( , ), pCDNA, CMV Fluc, (Promega), 5′UTR Fluc are from , MT1 is from , Untagged ΔCMV L1 was generated by NotI and SalI digest of JM101 L1.3, followed by gel extraction and purification of the L1-containing fragment. .. The purified L1-containing sequence was cloned into pBluescriptII (SK) digested with NotI and SalI.

Proliferation Assay:

Article Title: Mapping ER? Genomic Binding Sites Reveals Unique Genomic Features and Identifies EBF1 as an ER? Interactor
Article Snippet: .. Cell Proliferation Assay C4–12/Flag.ERβ cells were transfected in 24-well plates with pcDNA or EBF1 plasmids using FuGENE (Promega). .. Cell confluency, used to quantify cell growth, was measured in an Incucyte FLR live content imaging system (Essen Bioscience).

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    Promega pcdna sox9
    Luciferase assays of <t>Sox9</t> function on Spp1 activity. HEK293 (A) and MC-3T3 (B) cells were co-tranfected with 200 ng of the luciferase reporter construct pGL3 containing the Spp1 promoter region (no SRY binding sites) (pGL3- spp1 p), the 1599 bp 5’ Spp1 enhancer region containing SRY#1 (pGL3- spp1 En), or both the enhancer and promoter regions (pGL3- spp1 ), along with 200 ng of empty <t>pcDNA</t> (grey bars) or pcDNA containing full length murine Sox9 (pcDNA-Sox9, black bars). In addition, cells were co-transfected with 20 ng of pGL4 for normalization. Luciferase activity of pGL3- spp1 with empty pcDNA was set to 100%. Note repression of pGL3- spp1 activity (63%±8% (A) and 60.3%±12.6% (B) in the presence of pcDNA-Sox9. Cells were also co-transfected with pcDNA or pcDNA-Sox9 in the presence of pGL3- spp1 in which the SRY#1 had been mutated from TA CAAA G to TA GCAG G (pGL3- spp1 mut). Note that pcDNA-Sox9 could not significantly repress pGL3- spp1 mut unlike pGL3- spp1 . *Student's t−test
    Pcdna Sox9, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega mock pcdna mir nc
    miR-196a directly targets the HOXA5 gene. ( A ) A human HOXA5 or FOXO1 3’ untranslated region (UTR) fragment containing the wild-type or mutant miR-196a binding sequence was cloned downstream of the luciferase reporter gene. ( B ) The luciferase reporter plasmid containing wild-type or mutant HOXA5 and FOXO1 3’UTR were <t>co-transfected</t> into HEK-293 T cells with <t>pCDNA-miR-196a</t> or pCDNA-miR-NC. Luciferase activity was determined using the dual luciferase assay and shown as the relative firefly activity normalized to renilla activity. ( C ) qRT-PCR analyses of HOXA5 mRNA level following treatment of SPCA1 cells with miR-196a inhibitors or A549 cells with miR-196a mimics. ( D ) Western blot analyses of HOXA5 protein levels following treatment of SPCA1 cells with miR-196a inhibitors or A549 cells with miR-196a mimics. GAPDH was used as control. *P
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    Promega luciferase assay pcdna odd luc
    Effects of o -Phen, Zn-Phen, and Rh-Phen on prolyl hydroxylase domain-containing protein 2 (PHD2) activity in vascular endothelial cells. ( A ) Bovine aortic endothelial cells were transfected with <t>pcDNA-Luc</t> or <t>pcDNA-ODD-Luc</t> at 37 °C for 24 h. Amplified DNA products were separated by agarose gel electrophoresis and stained using ethidium bromide; ( B ) Bovine aortic endothelial cells were transfected with pcDNA-ODD-Luc at 37 °C for 12 h, treated with o -Phen, Zn-Phen, or Rh-Phen at 5 µM each at 37 °C for 24 h, and PHD2 activity was subsequently analyzed by luciferase assay. Values are means ± S.E.M. of five samples. ** p
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    Promega pcdna
    Fig. 2. ICSBP down-regulates PU.1 induced transactivation of the Dab2 promoter. ( A ) K562 cells were transfected with pGL3-Basic containing a 2 kb fragment of the mouse Dab2 promoter together with <t>pcDNA</t> or pcDNA-ICSBP and the reference vector <t>pRL-TK.</t> Luciferase reporter gene activities were measured as described in Materials and methods. ( B ) CV-1 cells were transiently transfected with a Dab2 promoter driven luciferase reporter gene (300 ng) along with the following expression vectors: PU.1 200 ng; ICSBP 200 ng or 600 ng. Empty vector DNA was added so that each reaction contained a total of 800 ng of expression plasmid. ( C ) Endogenously expressed PU.1 binds to the Dab2 regulatory sequence. K562 nuclear extracts were incubated with the immobilized Dab2 promoter DNA, and the bound fraction was analyzed by western blotting using antibodies as indicated. ( D ) ICSBP and PU.1 bind to the Dab2 promoter. Magnetic beads containing the 2 kb mouse Dab2 promoter were incubated with nuclear extracts of CV-1 cells, which were supplemented with 10 µl of 35 S-labeled ICSBP, PU.1, or both ICSBP and PU.1. The beads were washed and the eluates were subjected to SDS–PAGE and autoradiography. 35 S-labeled protein (1 µl) was used as an input control. Probing of the membrane with an anti-Stat3 antibody confirmed the specificity of the employed assay.
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    Image Search Results


    Luciferase assays of Sox9 function on Spp1 activity. HEK293 (A) and MC-3T3 (B) cells were co-tranfected with 200 ng of the luciferase reporter construct pGL3 containing the Spp1 promoter region (no SRY binding sites) (pGL3- spp1 p), the 1599 bp 5’ Spp1 enhancer region containing SRY#1 (pGL3- spp1 En), or both the enhancer and promoter regions (pGL3- spp1 ), along with 200 ng of empty pcDNA (grey bars) or pcDNA containing full length murine Sox9 (pcDNA-Sox9, black bars). In addition, cells were co-transfected with 20 ng of pGL4 for normalization. Luciferase activity of pGL3- spp1 with empty pcDNA was set to 100%. Note repression of pGL3- spp1 activity (63%±8% (A) and 60.3%±12.6% (B) in the presence of pcDNA-Sox9. Cells were also co-transfected with pcDNA or pcDNA-Sox9 in the presence of pGL3- spp1 in which the SRY#1 had been mutated from TA CAAA G to TA GCAG G (pGL3- spp1 mut). Note that pcDNA-Sox9 could not significantly repress pGL3- spp1 mut unlike pGL3- spp1 . *Student's t−test

    Journal: PLoS ONE

    Article Title: Sox9 Transcriptionally Represses Spp1 to Prevent Matrix Mineralization in Maturing Heart Valves and Chondrocytes

    doi: 10.1371/journal.pone.0026769

    Figure Lengend Snippet: Luciferase assays of Sox9 function on Spp1 activity. HEK293 (A) and MC-3T3 (B) cells were co-tranfected with 200 ng of the luciferase reporter construct pGL3 containing the Spp1 promoter region (no SRY binding sites) (pGL3- spp1 p), the 1599 bp 5’ Spp1 enhancer region containing SRY#1 (pGL3- spp1 En), or both the enhancer and promoter regions (pGL3- spp1 ), along with 200 ng of empty pcDNA (grey bars) or pcDNA containing full length murine Sox9 (pcDNA-Sox9, black bars). In addition, cells were co-transfected with 20 ng of pGL4 for normalization. Luciferase activity of pGL3- spp1 with empty pcDNA was set to 100%. Note repression of pGL3- spp1 activity (63%±8% (A) and 60.3%±12.6% (B) in the presence of pcDNA-Sox9. Cells were also co-transfected with pcDNA or pcDNA-Sox9 in the presence of pGL3- spp1 in which the SRY#1 had been mutated from TA CAAA G to TA GCAG G (pGL3- spp1 mut). Note that pcDNA-Sox9 could not significantly repress pGL3- spp1 mut unlike pGL3- spp1 . *Student's t−test

    Article Snippet: 200 ng of pGL3-spp1, pGL3-spp1 mut or pGL3 (200 ng/well), and pcDNA or pcDNA-Sox9 (200 ng/well), were co-transfected into each well, along with 20 ng of pGL4 (Renilla luciferase, Promega).

    Techniques: Luciferase, Activity Assay, Construct, Binding Assay, Transfection

    miR-196a directly targets the HOXA5 gene. ( A ) A human HOXA5 or FOXO1 3’ untranslated region (UTR) fragment containing the wild-type or mutant miR-196a binding sequence was cloned downstream of the luciferase reporter gene. ( B ) The luciferase reporter plasmid containing wild-type or mutant HOXA5 and FOXO1 3’UTR were co-transfected into HEK-293 T cells with pCDNA-miR-196a or pCDNA-miR-NC. Luciferase activity was determined using the dual luciferase assay and shown as the relative firefly activity normalized to renilla activity. ( C ) qRT-PCR analyses of HOXA5 mRNA level following treatment of SPCA1 cells with miR-196a inhibitors or A549 cells with miR-196a mimics. ( D ) Western blot analyses of HOXA5 protein levels following treatment of SPCA1 cells with miR-196a inhibitors or A549 cells with miR-196a mimics. GAPDH was used as control. *P

    Journal: BMC Cancer

    Article Title: MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5

    doi: 10.1186/1471-2407-12-348

    Figure Lengend Snippet: miR-196a directly targets the HOXA5 gene. ( A ) A human HOXA5 or FOXO1 3’ untranslated region (UTR) fragment containing the wild-type or mutant miR-196a binding sequence was cloned downstream of the luciferase reporter gene. ( B ) The luciferase reporter plasmid containing wild-type or mutant HOXA5 and FOXO1 3’UTR were co-transfected into HEK-293 T cells with pCDNA-miR-196a or pCDNA-miR-NC. Luciferase activity was determined using the dual luciferase assay and shown as the relative firefly activity normalized to renilla activity. ( C ) qRT-PCR analyses of HOXA5 mRNA level following treatment of SPCA1 cells with miR-196a inhibitors or A549 cells with miR-196a mimics. ( D ) Western blot analyses of HOXA5 protein levels following treatment of SPCA1 cells with miR-196a inhibitors or A549 cells with miR-196a mimics. GAPDH was used as control. *P

    Article Snippet: Human HEK293T cells grown in a 48-well plate were co-transfected with 200 ng of either mock pCDNA/miR-NC or pCDNA/miR-196a, 10 ng of firefly luciferase reporter comprising wild type or mutant 3’UTR of HOXA5 gene, and 2 ng of pRL-TK (Promega, Madison, WI, USA) using Lipofectamie 2000 ( Invitrogen, USA).

    Techniques: Mutagenesis, Binding Assay, Sequencing, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Quantitative RT-PCR, Western Blot

    Effect of miR-196a on cell proliferation in vitro. ( A , B ) SPC-A1 cells were transfected with miR-196a inhibitors or anti-miR-NC, and A549 cells were transfected with pCDNA/miR-196a or pCDNA/miR-NC. MTT assay was performed to determine the proliferation of SPC-A1 or A549 cells. Data represent the mean ± S.D. from three independent experiments. ( C , D ) Colony-forming growth assays were performed to determine the proliferation of SPC-A1 or A549 cells. The colonies were counted and captured. *P

    Journal: BMC Cancer

    Article Title: MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5

    doi: 10.1186/1471-2407-12-348

    Figure Lengend Snippet: Effect of miR-196a on cell proliferation in vitro. ( A , B ) SPC-A1 cells were transfected with miR-196a inhibitors or anti-miR-NC, and A549 cells were transfected with pCDNA/miR-196a or pCDNA/miR-NC. MTT assay was performed to determine the proliferation of SPC-A1 or A549 cells. Data represent the mean ± S.D. from three independent experiments. ( C , D ) Colony-forming growth assays were performed to determine the proliferation of SPC-A1 or A549 cells. The colonies were counted and captured. *P

    Article Snippet: Human HEK293T cells grown in a 48-well plate were co-transfected with 200 ng of either mock pCDNA/miR-NC or pCDNA/miR-196a, 10 ng of firefly luciferase reporter comprising wild type or mutant 3’UTR of HOXA5 gene, and 2 ng of pRL-TK (Promega, Madison, WI, USA) using Lipofectamie 2000 ( Invitrogen, USA).

    Techniques: In Vitro, Transfection, MTT Assay

    qRT-PCR analysis of miR-196a in NSCLC tissues and matched normal tissues. ( A ) Determining miR-196a expression in NSCLC tissues and its clinical significance. miR-196a was detected in 34 pairs of NSCLC tissues by qRT-PCR. Data are presented as fold-changes in tumor tissues relative to normal tissues. miR-196a expression was significantly higher in patients with a higher pathological stage and lymph-node metastasis. ( B ) Analysis of miR-196a expression levels in NSCLC cell lines (A549, SPC-A1, NCI-H1650, NCI-H1299 and SK-MES-1) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( C ) Analysis of miR-196a expression following treatment of SPCA1 cells with anti-miR-NC or miR-196a inhibitors by qRT-PCR. ( D ) Analysis of miR-196a expression following treatment of A549 cells with miR-NC, miR-196a mimics, pCDNA-NC or pCDNA-miR-196a by qRT-PCR. All experiments were performed in biological triplicate with three technical replicates. *P

    Journal: BMC Cancer

    Article Title: MicroRNA-196a promotes non-small cell lung cancer cell proliferation and invasion through targeting HOXA5

    doi: 10.1186/1471-2407-12-348

    Figure Lengend Snippet: qRT-PCR analysis of miR-196a in NSCLC tissues and matched normal tissues. ( A ) Determining miR-196a expression in NSCLC tissues and its clinical significance. miR-196a was detected in 34 pairs of NSCLC tissues by qRT-PCR. Data are presented as fold-changes in tumor tissues relative to normal tissues. miR-196a expression was significantly higher in patients with a higher pathological stage and lymph-node metastasis. ( B ) Analysis of miR-196a expression levels in NSCLC cell lines (A549, SPC-A1, NCI-H1650, NCI-H1299 and SK-MES-1) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( C ) Analysis of miR-196a expression following treatment of SPCA1 cells with anti-miR-NC or miR-196a inhibitors by qRT-PCR. ( D ) Analysis of miR-196a expression following treatment of A549 cells with miR-NC, miR-196a mimics, pCDNA-NC or pCDNA-miR-196a by qRT-PCR. All experiments were performed in biological triplicate with three technical replicates. *P

    Article Snippet: Human HEK293T cells grown in a 48-well plate were co-transfected with 200 ng of either mock pCDNA/miR-NC or pCDNA/miR-196a, 10 ng of firefly luciferase reporter comprising wild type or mutant 3’UTR of HOXA5 gene, and 2 ng of pRL-TK (Promega, Madison, WI, USA) using Lipofectamie 2000 ( Invitrogen, USA).

    Techniques: Quantitative RT-PCR, Expressing

    Effects of o -Phen, Zn-Phen, and Rh-Phen on prolyl hydroxylase domain-containing protein 2 (PHD2) activity in vascular endothelial cells. ( A ) Bovine aortic endothelial cells were transfected with pcDNA-Luc or pcDNA-ODD-Luc at 37 °C for 24 h. Amplified DNA products were separated by agarose gel electrophoresis and stained using ethidium bromide; ( B ) Bovine aortic endothelial cells were transfected with pcDNA-ODD-Luc at 37 °C for 12 h, treated with o -Phen, Zn-Phen, or Rh-Phen at 5 µM each at 37 °C for 24 h, and PHD2 activity was subsequently analyzed by luciferase assay. Values are means ± S.E.M. of five samples. ** p

    Journal: International Journal of Molecular Sciences

    Article Title: Induction of Syndecan-4 by Organic–Inorganic Hybrid Molecules with a 1,10-Phenanthroline Structure in Cultured Vascular Endothelial Cells

    doi: 10.3390/ijms18020352

    Figure Lengend Snippet: Effects of o -Phen, Zn-Phen, and Rh-Phen on prolyl hydroxylase domain-containing protein 2 (PHD2) activity in vascular endothelial cells. ( A ) Bovine aortic endothelial cells were transfected with pcDNA-Luc or pcDNA-ODD-Luc at 37 °C for 24 h. Amplified DNA products were separated by agarose gel electrophoresis and stained using ethidium bromide; ( B ) Bovine aortic endothelial cells were transfected with pcDNA-ODD-Luc at 37 °C for 12 h, treated with o -Phen, Zn-Phen, or Rh-Phen at 5 µM each at 37 °C for 24 h, and PHD2 activity was subsequently analyzed by luciferase assay. Values are means ± S.E.M. of five samples. ** p

    Article Snippet: Luciferase Assay pcDNA-ODD-Luc and the Renilla luciferase-expression plasmid pRL-SV40 (Promega) were transfected into vascular endothelial cells using Lipofectamine LTX and PLUS reagents according to manufacturer instructions.

    Techniques: Activity Assay, Transfection, Amplification, Agarose Gel Electrophoresis, Staining, Luciferase

    Fig. 2. ICSBP down-regulates PU.1 induced transactivation of the Dab2 promoter. ( A ) K562 cells were transfected with pGL3-Basic containing a 2 kb fragment of the mouse Dab2 promoter together with pcDNA or pcDNA-ICSBP and the reference vector pRL-TK. Luciferase reporter gene activities were measured as described in Materials and methods. ( B ) CV-1 cells were transiently transfected with a Dab2 promoter driven luciferase reporter gene (300 ng) along with the following expression vectors: PU.1 200 ng; ICSBP 200 ng or 600 ng. Empty vector DNA was added so that each reaction contained a total of 800 ng of expression plasmid. ( C ) Endogenously expressed PU.1 binds to the Dab2 regulatory sequence. K562 nuclear extracts were incubated with the immobilized Dab2 promoter DNA, and the bound fraction was analyzed by western blotting using antibodies as indicated. ( D ) ICSBP and PU.1 bind to the Dab2 promoter. Magnetic beads containing the 2 kb mouse Dab2 promoter were incubated with nuclear extracts of CV-1 cells, which were supplemented with 10 µl of 35 S-labeled ICSBP, PU.1, or both ICSBP and PU.1. The beads were washed and the eluates were subjected to SDS–PAGE and autoradiography. 35 S-labeled protein (1 µl) was used as an input control. Probing of the membrane with an anti-Stat3 antibody confirmed the specificity of the employed assay.

    Journal: The EMBO Journal

    Article Title: Disabled-2 is transcriptionally regulated by ICSBP and augments macrophage spreading and adhesion

    doi: 10.1093/emboj/21.3.211

    Figure Lengend Snippet: Fig. 2. ICSBP down-regulates PU.1 induced transactivation of the Dab2 promoter. ( A ) K562 cells were transfected with pGL3-Basic containing a 2 kb fragment of the mouse Dab2 promoter together with pcDNA or pcDNA-ICSBP and the reference vector pRL-TK. Luciferase reporter gene activities were measured as described in Materials and methods. ( B ) CV-1 cells were transiently transfected with a Dab2 promoter driven luciferase reporter gene (300 ng) along with the following expression vectors: PU.1 200 ng; ICSBP 200 ng or 600 ng. Empty vector DNA was added so that each reaction contained a total of 800 ng of expression plasmid. ( C ) Endogenously expressed PU.1 binds to the Dab2 regulatory sequence. K562 nuclear extracts were incubated with the immobilized Dab2 promoter DNA, and the bound fraction was analyzed by western blotting using antibodies as indicated. ( D ) ICSBP and PU.1 bind to the Dab2 promoter. Magnetic beads containing the 2 kb mouse Dab2 promoter were incubated with nuclear extracts of CV-1 cells, which were supplemented with 10 µl of 35 S-labeled ICSBP, PU.1, or both ICSBP and PU.1. The beads were washed and the eluates were subjected to SDS–PAGE and autoradiography. 35 S-labeled protein (1 µl) was used as an input control. Probing of the membrane with an anti-Stat3 antibody confirmed the specificity of the employed assay.

    Article Snippet: Vectors pRL-Null, pRL-TK, pcDNA and pGL3-basic were purchased from Promega.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Expressing, Sequencing, Incubation, Western Blot, Magnetic Beads, Labeling, SDS Page, Autoradiography