Structured Review

TaKaRa pegfp c1
Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected <t>pEGFP-C1</t> (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
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Images

1) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

2) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

3) Product Images from "Caspase-Dependent Regulation and Subcellular Redistribution of the Transcriptional Modulator YY1 during Apoptosis"

Article Title: Caspase-Dependent Regulation and Subcellular Redistribution of the Transcriptional Modulator YY1 during Apoptosis

Journal:

doi: 10.1128/MCB.25.9.3704-3714.2005

Enhancement of apoptosis by YY1Δ119. (A) HeLa cells were cotransfected with pEGFP-C1 (0.05 μg) in order to detect the transfected cells, an expression plasmid for the death receptor Fas (pCR3-Fas; 0.1 μg), and plasmids expressing
Figure Legend Snippet: Enhancement of apoptosis by YY1Δ119. (A) HeLa cells were cotransfected with pEGFP-C1 (0.05 μg) in order to detect the transfected cells, an expression plasmid for the death receptor Fas (pCR3-Fas; 0.1 μg), and plasmids expressing

Techniques Used: Transfection, Expressing, Plasmid Preparation

4) Product Images from "The Open Reading Frame 57 Gene Product of Herpesvirus Saimiri Shuttles between the Nucleus and Cytoplasm and Is Involved in Viral RNA Nuclear Export"

Article Title: The Open Reading Frame 57 Gene Product of Herpesvirus Saimiri Shuttles between the Nucleus and Cytoplasm and Is Involved in Viral RNA Nuclear Export

Journal: Journal of Virology

doi:

The ORF 57 gene product expresses a nuclear export signal. Cos-7 cell monolayers were transfected with 2 μg of either pEGFP-C1 (i) or pEGFP-57NES (ii). After 24 h, the subcellular localization of GFP was observed by using fluorescence microscopy.
Figure Legend Snippet: The ORF 57 gene product expresses a nuclear export signal. Cos-7 cell monolayers were transfected with 2 μg of either pEGFP-C1 (i) or pEGFP-57NES (ii). After 24 h, the subcellular localization of GFP was observed by using fluorescence microscopy.

Techniques Used: Transfection, Fluorescence, Microscopy

5) Product Images from "The anti-apoptotic PON2 protein is Wnt/β-catenin-regulated and correlates with radiotherapy resistance in OSCC patients"

Article Title: The anti-apoptotic PON2 protein is Wnt/β-catenin-regulated and correlates with radiotherapy resistance in OSCC patients

Journal: Oncotarget

doi: 10.18632/oncotarget.9013

PON2 expression is regulated by LEF-1 and TCF4, but not TCF1 K562 cells were co-transfected with plasmids encoding a firefly luciferase gene under the expressional control of the 7-LEF-fos-luc A. or PON2 promoter fragment B. , a plasmid for constitutive expression of Renilla luciferase (normalization) and pEGFP-C1, pEGFP-C1-dnLEF-1, pEGFP-C1-TCF1 or pEGFP-C1-TCF4 plasmids. At 4 h after transfection, cells were treated with SB216763 (25 μM) for 24 h. Subsequently 7-LEF-fos-luc or PON2 promoter induction was analyzed, normalized to Renilla luciferase activity and expressed as fold induction. Symbols represent mean ± S.E.M. n = 2; * P
Figure Legend Snippet: PON2 expression is regulated by LEF-1 and TCF4, but not TCF1 K562 cells were co-transfected with plasmids encoding a firefly luciferase gene under the expressional control of the 7-LEF-fos-luc A. or PON2 promoter fragment B. , a plasmid for constitutive expression of Renilla luciferase (normalization) and pEGFP-C1, pEGFP-C1-dnLEF-1, pEGFP-C1-TCF1 or pEGFP-C1-TCF4 plasmids. At 4 h after transfection, cells were treated with SB216763 (25 μM) for 24 h. Subsequently 7-LEF-fos-luc or PON2 promoter induction was analyzed, normalized to Renilla luciferase activity and expressed as fold induction. Symbols represent mean ± S.E.M. n = 2; * P

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

6) Product Images from "GENE THERAPY OF CARCINOMA USING ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION"

Article Title: GENE THERAPY OF CARCINOMA USING ULTRASOUND-TARGETED MICROBUBBLE DESTRUCTION

Journal: Ultrasound in medicine & biology

doi: 10.1016/j.ultrasmedbio.2010.11.011

Postmortem histology of tumors after intravenous delivery of either pCMV-TK (a, c, d) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV. (a, b) Acellular zones ( arrows ) visible under H E stain. (c) Merged image of two sister
Figure Legend Snippet: Postmortem histology of tumors after intravenous delivery of either pCMV-TK (a, c, d) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV. (a, b) Acellular zones ( arrows ) visible under H E stain. (c) Merged image of two sister

Techniques Used: Staining

Immunofluorescent staining of GFP in murine tumors 3 d after intravenous delivery of pEGFP-C1 bound to microbubbles and treatment with ultrasound (a, b) or no ultrasound (c). GFP-positive staining was seen both in hollow structures (a) as well as individual
Figure Legend Snippet: Immunofluorescent staining of GFP in murine tumors 3 d after intravenous delivery of pEGFP-C1 bound to microbubbles and treatment with ultrasound (a, b) or no ultrasound (c). GFP-positive staining was seen both in hollow structures (a) as well as individual

Techniques Used: Staining

Postmortem TUNEL assays from murine tumors after intravenous injection of either pCMV-TK (a) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV.
Figure Legend Snippet: Postmortem TUNEL assays from murine tumors after intravenous injection of either pCMV-TK (a) or pEGFP-C1 (b)-loaded microbubbles and treatment with ultrasound and GCV.

Techniques Used: TUNEL Assay, Injection

Growth of murine tumors after intravenous injection of either pCMV-TK (□) or pEGFP-C1 (▲)-loaded microbubbles and treated with ultrasound. Daily GCV injections began on day 3. Best-fit lines were calculated from all data points in each
Figure Legend Snippet: Growth of murine tumors after intravenous injection of either pCMV-TK (□) or pEGFP-C1 (▲)-loaded microbubbles and treated with ultrasound. Daily GCV injections began on day 3. Best-fit lines were calculated from all data points in each

Techniques Used: Injection

7) Product Images from "Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus"

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054075

Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).
Figure Legend Snippet: Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).

Techniques Used: Transfection

Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.
Figure Legend Snippet: Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.

Techniques Used: Immunofluorescence, Transfection, Incubation, Laser-Scanning Microscopy, Staining

8) Product Images from "RACK1 is involved in endothelial barrier regulation via its two novel interacting partners"

Article Title: RACK1 is involved in endothelial barrier regulation via its two novel interacting partners

Journal: Cell Communication and Signaling : CCS

doi: 10.1186/1478-811X-11-2

RACK1 interacts with PP1cδ via TIMAP. ( A ): Bacterially expressed glutathione S-transferase (GST) and GST-tagged wild-type TIMAP were loaded onto glutathione-Sepharose as described in Materials and Methods. After a washing step the resin samples were incubated with BPAEC lysate (CL) or cell lysis buffer (LB). Non-binding proteins were washed out and the bound proteins were eluted with 10 mM glutathion. Western blot probed with RACK1 specific antibody ( A ) of the endothelial cell lysate (CL) and the eluted fractions after the pull-down are shown. ( B , C ): RACK1 or TIMAP was immunoprecipitated from lysates of BPAEC ( B ) and BPAEC or HeLa ( C ) cells as described in Materials and Methods. IP complexes were probed for TIMAP and RACK1 ( B ) or PP1cδ ( C ). CL: cell lysate, Ø AB: control of IP from BPAEC without the addition of antibody. ( D , E ): RACK1 or GFP was immunoprecipitated from non transfected (CL), non-siRNA, TIMAP specific siRNA (si TIMAP), pEGFP-C1 (GFP), pEGFP-C1 TIMAP WT (GFP-Twt) or pEGFP-C1 TIMAPΔpp1c (GFP-TΔ) transfected HeLa cell lysates. IP complexes were probed for GFP, RACK1 or PP1cδ.
Figure Legend Snippet: RACK1 interacts with PP1cδ via TIMAP. ( A ): Bacterially expressed glutathione S-transferase (GST) and GST-tagged wild-type TIMAP were loaded onto glutathione-Sepharose as described in Materials and Methods. After a washing step the resin samples were incubated with BPAEC lysate (CL) or cell lysis buffer (LB). Non-binding proteins were washed out and the bound proteins were eluted with 10 mM glutathion. Western blot probed with RACK1 specific antibody ( A ) of the endothelial cell lysate (CL) and the eluted fractions after the pull-down are shown. ( B , C ): RACK1 or TIMAP was immunoprecipitated from lysates of BPAEC ( B ) and BPAEC or HeLa ( C ) cells as described in Materials and Methods. IP complexes were probed for TIMAP and RACK1 ( B ) or PP1cδ ( C ). CL: cell lysate, Ø AB: control of IP from BPAEC without the addition of antibody. ( D , E ): RACK1 or GFP was immunoprecipitated from non transfected (CL), non-siRNA, TIMAP specific siRNA (si TIMAP), pEGFP-C1 (GFP), pEGFP-C1 TIMAP WT (GFP-Twt) or pEGFP-C1 TIMAPΔpp1c (GFP-TΔ) transfected HeLa cell lysates. IP complexes were probed for GFP, RACK1 or PP1cδ.

Techniques Used: Incubation, Lysis, Binding Assay, Western Blot, Immunoprecipitation, Transfection

9) Product Images from "Cellular Protein HAX1 Interacts with the Influenza A Virus PA Polymerase Subunit and Impedes Its Nuclear Translocation"

Article Title: Cellular Protein HAX1 Interacts with the Influenza A Virus PA Polymerase Subunit and Impedes Its Nuclear Translocation

Journal: Journal of Virology

doi: 10.1128/JVI.00939-12

) or vector plasmid pEGFP-C1.
Figure Legend Snippet: ) or vector plasmid pEGFP-C1.

Techniques Used: Plasmid Preparation

10) Product Images from "The Ras/Raf/ERK signalling pathway drives Schwann cell dedifferentiation"

Article Title: The Ras/Raf/ERK signalling pathway drives Schwann cell dedifferentiation

Journal: The EMBO Journal

doi: 10.1038/sj.emboj.7600309

Oncogenic Ras blocks Schwann cell differentiation and can induce dedifferentiation. ( A ) NS cells infected with retroviruses expressing oncogenic Ras LXSN (Ras) or vector alone (LXSN) were treated with db-cAMP in defined medium for 3 days prior to analysis by Western blotting (i) or real-time PCR (ii). The real-time PCR data shown have been equalised for GAPDH levels. ( B ) Freshly prepared NS cells treated with db-cAMP for 2 days were microinjected with H-RasVal12 or empty vector control, together with pEGFP-C1. Cultures were fixed after 48 h and analysed by immunofluorescence. (i, ii) Quantification of results showing the percentage of GFP-labelled periaxin-, P0-, Krox-20- and cyclin D1-positive cells and s.d. for each experimental condition. (iii) An example of the results obtained using immunofluorescence against periaxin (red), GFP (green) and with Hoechst (blue) to visualise DNA. (The larger nuclei belong to fibroblasts contaminating the primary Schwann cell cultures.) The bottom panels show triple labelling with the same field shown in the top panels without the GFP channel, in order to enable clear identification of periaxin-positive cells.
Figure Legend Snippet: Oncogenic Ras blocks Schwann cell differentiation and can induce dedifferentiation. ( A ) NS cells infected with retroviruses expressing oncogenic Ras LXSN (Ras) or vector alone (LXSN) were treated with db-cAMP in defined medium for 3 days prior to analysis by Western blotting (i) or real-time PCR (ii). The real-time PCR data shown have been equalised for GAPDH levels. ( B ) Freshly prepared NS cells treated with db-cAMP for 2 days were microinjected with H-RasVal12 or empty vector control, together with pEGFP-C1. Cultures were fixed after 48 h and analysed by immunofluorescence. (i, ii) Quantification of results showing the percentage of GFP-labelled periaxin-, P0-, Krox-20- and cyclin D1-positive cells and s.d. for each experimental condition. (iii) An example of the results obtained using immunofluorescence against periaxin (red), GFP (green) and with Hoechst (blue) to visualise DNA. (The larger nuclei belong to fibroblasts contaminating the primary Schwann cell cultures.) The bottom panels show triple labelling with the same field shown in the top panels without the GFP channel, in order to enable clear identification of periaxin-positive cells.

Techniques Used: Cell Differentiation, Infection, Expressing, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Immunofluorescence

11) Product Images from "SOX2 suppresses the mobility of urothelial carcinoma by promoting the expression of S100A14"

Article Title: SOX2 suppresses the mobility of urothelial carcinoma by promoting the expression of S100A14

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.06.016

Suppression of SOX2 leads to an increase in the growth and mobility of BFTC905 cells. (A) The day after seeding the BFTC905/shLuc and BFTC905/shSOX2 cells was designated as day 0. The cell density of an initial 1×106 cells was determined by crystal violet staining. The cultures with 5×10 5 , 2.5×10 5 , or 1.25×105 cells were allowed to grow for additional one, two, and three days, respectively. At each time point, the cell densities were determined by crystal violet staining, and the readings were normalized by multiplying the value with the number of respective days. The relative increase in the cell density was calculated by dividing the normalized crystal violet reading with the reading of the day 0 culture. Student's t -test was carried out to determine the statistical significance of the differential growth at each point. The p value smaller than 0.05 is represented as an asterisk. (B) The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were created by transfecting the BFTC905/shLuc and BFTC905/shSOX2 cells with pTag-RFP-N and pEGFP-C1, respectively. After two weeks of antibiotic selection, the cells expressing the fluorescent proteins were collected using a BD FACS Aria III cell sorter. The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were then mixed together and cultured in a glass-bottom dish under normal growth and passage conditions for 15 days. At days 0 and 15, the cells were imaged using an Olympus laser scanning confocal microscope. (C) The percentage of BFTC905/shSOX2-EGFP cells in the population was determined by counting more than 200 cells from 10 random images. The results of three independent experiments are shown. (D) The BFTC905/shLuc and BFTC905/shSOX2 cells were seeded in the cell inserts for 24 h. After the inserts were removed, the cells were cultured in medium containing 1% fetal bovine serum for 24 h. The images were captured with a differential interference contrast-equipped microscope at 0 and 24 h. (E) Equal numbers of BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were seeded into the upper chamber of the transwell apparatus. After attachment, the cells were allowed to migrate in the absence of serum for 24 h. The total cells were first imaged using a fluorescence microscope (panels A–C). After removing the non-migrating cells on the upper side of the transwell membrane, the remaining cells on the lower side were imaged using an Olympus laser scanning confocal microscope (panels D– F).
Figure Legend Snippet: Suppression of SOX2 leads to an increase in the growth and mobility of BFTC905 cells. (A) The day after seeding the BFTC905/shLuc and BFTC905/shSOX2 cells was designated as day 0. The cell density of an initial 1×106 cells was determined by crystal violet staining. The cultures with 5×10 5 , 2.5×10 5 , or 1.25×105 cells were allowed to grow for additional one, two, and three days, respectively. At each time point, the cell densities were determined by crystal violet staining, and the readings were normalized by multiplying the value with the number of respective days. The relative increase in the cell density was calculated by dividing the normalized crystal violet reading with the reading of the day 0 culture. Student's t -test was carried out to determine the statistical significance of the differential growth at each point. The p value smaller than 0.05 is represented as an asterisk. (B) The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were created by transfecting the BFTC905/shLuc and BFTC905/shSOX2 cells with pTag-RFP-N and pEGFP-C1, respectively. After two weeks of antibiotic selection, the cells expressing the fluorescent proteins were collected using a BD FACS Aria III cell sorter. The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were then mixed together and cultured in a glass-bottom dish under normal growth and passage conditions for 15 days. At days 0 and 15, the cells were imaged using an Olympus laser scanning confocal microscope. (C) The percentage of BFTC905/shSOX2-EGFP cells in the population was determined by counting more than 200 cells from 10 random images. The results of three independent experiments are shown. (D) The BFTC905/shLuc and BFTC905/shSOX2 cells were seeded in the cell inserts for 24 h. After the inserts were removed, the cells were cultured in medium containing 1% fetal bovine serum for 24 h. The images were captured with a differential interference contrast-equipped microscope at 0 and 24 h. (E) Equal numbers of BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were seeded into the upper chamber of the transwell apparatus. After attachment, the cells were allowed to migrate in the absence of serum for 24 h. The total cells were first imaged using a fluorescence microscope (panels A–C). After removing the non-migrating cells on the upper side of the transwell membrane, the remaining cells on the lower side were imaged using an Olympus laser scanning confocal microscope (panels D– F).

Techniques Used: Staining, Selection, Expressing, FACS, Cell Culture, Microscopy, Fluorescence

12) Product Images from "SOX2 suppresses the mobility of urothelial carcinoma by promoting the expression of S100A14"

Article Title: SOX2 suppresses the mobility of urothelial carcinoma by promoting the expression of S100A14

Journal: Biochemistry and Biophysics Reports

doi: 10.1016/j.bbrep.2016.06.016

Suppression of SOX2 leads to an increase in the growth and mobility of BFTC905 cells. (A) The day after seeding the BFTC905/shLuc and BFTC905/shSOX2 cells was designated as day 0. The cell density of an initial 1×106 cells was determined by crystal violet staining. The cultures with 5×10 5 , 2.5×10 5 , or 1.25×105 cells were allowed to grow for additional one, two, and three days, respectively. At each time point, the cell densities were determined by crystal violet staining, and the readings were normalized by multiplying the value with the number of respective days. The relative increase in the cell density was calculated by dividing the normalized crystal violet reading with the reading of the day 0 culture. Student's t -test was carried out to determine the statistical significance of the differential growth at each point. The p value smaller than 0.05 is represented as an asterisk. (B) The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were created by transfecting the BFTC905/shLuc and BFTC905/shSOX2 cells with pTag-RFP-N and pEGFP-C1, respectively. After two weeks of antibiotic selection, the cells expressing the fluorescent proteins were collected using a BD FACS Aria III cell sorter. The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were then mixed together and cultured in a glass-bottom dish under normal growth and passage conditions for 15 days. At days 0 and 15, the cells were imaged using an Olympus laser scanning confocal microscope. (C) The percentage of BFTC905/shSOX2-EGFP cells in the population was determined by counting more than 200 cells from 10 random images. The results of three independent experiments are shown. (D) The BFTC905/shLuc and BFTC905/shSOX2 cells were seeded in the cell inserts for 24 h. After the inserts were removed, the cells were cultured in medium containing 1% fetal bovine serum for 24 h. The images were captured with a differential interference contrast-equipped microscope at 0 and 24 h. (E) Equal numbers of BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were seeded into the upper chamber of the transwell apparatus. After attachment, the cells were allowed to migrate in the absence of serum for 24 h. The total cells were first imaged using a fluorescence microscope (panels A–C). After removing the non-migrating cells on the upper side of the transwell membrane, the remaining cells on the lower side were imaged using an Olympus laser scanning confocal microscope (panels D– F).
Figure Legend Snippet: Suppression of SOX2 leads to an increase in the growth and mobility of BFTC905 cells. (A) The day after seeding the BFTC905/shLuc and BFTC905/shSOX2 cells was designated as day 0. The cell density of an initial 1×106 cells was determined by crystal violet staining. The cultures with 5×10 5 , 2.5×10 5 , or 1.25×105 cells were allowed to grow for additional one, two, and three days, respectively. At each time point, the cell densities were determined by crystal violet staining, and the readings were normalized by multiplying the value with the number of respective days. The relative increase in the cell density was calculated by dividing the normalized crystal violet reading with the reading of the day 0 culture. Student's t -test was carried out to determine the statistical significance of the differential growth at each point. The p value smaller than 0.05 is represented as an asterisk. (B) The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were created by transfecting the BFTC905/shLuc and BFTC905/shSOX2 cells with pTag-RFP-N and pEGFP-C1, respectively. After two weeks of antibiotic selection, the cells expressing the fluorescent proteins were collected using a BD FACS Aria III cell sorter. The BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were then mixed together and cultured in a glass-bottom dish under normal growth and passage conditions for 15 days. At days 0 and 15, the cells were imaged using an Olympus laser scanning confocal microscope. (C) The percentage of BFTC905/shSOX2-EGFP cells in the population was determined by counting more than 200 cells from 10 random images. The results of three independent experiments are shown. (D) The BFTC905/shLuc and BFTC905/shSOX2 cells were seeded in the cell inserts for 24 h. After the inserts were removed, the cells were cultured in medium containing 1% fetal bovine serum for 24 h. The images were captured with a differential interference contrast-equipped microscope at 0 and 24 h. (E) Equal numbers of BFTC905/shLuc-RFP and BFTC905/shSOX2-EGFP cells were seeded into the upper chamber of the transwell apparatus. After attachment, the cells were allowed to migrate in the absence of serum for 24 h. The total cells were first imaged using a fluorescence microscope (panels A–C). After removing the non-migrating cells on the upper side of the transwell membrane, the remaining cells on the lower side were imaged using an Olympus laser scanning confocal microscope (panels D– F).

Techniques Used: Staining, Selection, Expressing, FACS, Cell Culture, Microscopy, Fluorescence

13) Product Images from "Otud7a Knockout Mice Recapitulate Many Neurological Features of 15q13.3 Microdeletion Syndrome"

Article Title: Otud7a Knockout Mice Recapitulate Many Neurological Features of 15q13.3 Microdeletion Syndrome

Journal: American Journal of Human Genetics

doi: 10.1016/j.ajhg.2018.01.005

OTUD7A Localizes to Dendritic Spines and Regulates Spine Density (A) OTUD7A localizes to dendritic spines. Mouse primary cortical cultures were co-transfected with pEGFP-c1 and 3xFLAG-OTUD7A on DIV 7. On DIV 14, immunofluorescent imaging was performed, with GFP in green (Alexa488) and FLAG in red (Cy3). The bottom right corner of each image shows a magnification of the inset box. (B) Primary cortical neurons from Otud7a -null mice show decreased total spine density compared to wild-type neurons, which are rescued by overexpression of Otud7a . To compare between genotypes, primary cortical neurons from Otud7a -null mice or wild-type littermates were transfected with pEGFP-c1 and stained with GFP antibody. As a rescue experiment, primary neurons from Otud7a -null mice were transfected with 700 ng 3xFLAG-OTUD7A per 24-well. We investigated 42 neurons/dendrites from 9 null mice, 41 neurons/dendrites from 3 wild-type mice, and 31 neurons/dendrites from 9 null mice in the rescue experiments. Top: representative examples of dendritic segments of the immune-stained neurons. Bottom: quantification of dendritic spines. Statistical significance was assessed using two-tailed t test. Data are presented as mean ± SEM ( ∗ p
Figure Legend Snippet: OTUD7A Localizes to Dendritic Spines and Regulates Spine Density (A) OTUD7A localizes to dendritic spines. Mouse primary cortical cultures were co-transfected with pEGFP-c1 and 3xFLAG-OTUD7A on DIV 7. On DIV 14, immunofluorescent imaging was performed, with GFP in green (Alexa488) and FLAG in red (Cy3). The bottom right corner of each image shows a magnification of the inset box. (B) Primary cortical neurons from Otud7a -null mice show decreased total spine density compared to wild-type neurons, which are rescued by overexpression of Otud7a . To compare between genotypes, primary cortical neurons from Otud7a -null mice or wild-type littermates were transfected with pEGFP-c1 and stained with GFP antibody. As a rescue experiment, primary neurons from Otud7a -null mice were transfected with 700 ng 3xFLAG-OTUD7A per 24-well. We investigated 42 neurons/dendrites from 9 null mice, 41 neurons/dendrites from 3 wild-type mice, and 31 neurons/dendrites from 9 null mice in the rescue experiments. Top: representative examples of dendritic segments of the immune-stained neurons. Bottom: quantification of dendritic spines. Statistical significance was assessed using two-tailed t test. Data are presented as mean ± SEM ( ∗ p

Techniques Used: Transfection, Imaging, Mouse Assay, Over Expression, Staining, Two Tailed Test

14) Product Images from "Characterization of the Expression, Intracellular Localization, and Replication Complex Association of the Putative Mouse Hepatitis Virus RNA-Dependent RNA Polymerase"

Article Title: Characterization of the Expression, Intracellular Localization, and Replication Complex Association of the Putative Mouse Hepatitis Virus RNA-Dependent RNA Polymerase

Journal: Journal of Virology

doi: 10.1128/JVI.77.19.10515-10527.2003

Targeting of Gpol to replication complexes during MHV infection. (A) Cloning of MHV Pol. The pol region of gene 1 was cloned by RT-PCR. Primer-generated substitutions were made to nucleotides in both the slippery sequence (boldfaced) and the pseudoknot in order to eliminate the potential for a −1 ribosomal frameshift while maintaining the coding sequence of pol . Mutated residues are shown in red, and the resulting amino acid sequences for both wild-type Pol and cloned Pol are given above nucleotide codons. pol cDNA was subcloned into pEGFP-C1 and expressed as a fusion to the carboxy terminus of GFP (Gpol). (B and C) DBT cells on glass coverslips were transfected with cDNA encoding either GFP or Gpol. Twenty-four hours posttransfection, cells were either infected with MHV or mock infected for 5.5 h, fixed, and either imaged for GFP fluorescence (B) or subjected to indirect immunofluorescence as described in Materials and Methods by using antibodies against the MHV structural proteins M and N (C). Multinucleated cells are a cytopathic effect of viral infection. In all images, GFP is shown as green and antibody staining is shown as red. Merged images are shown with areas of colocalization in yellow.
Figure Legend Snippet: Targeting of Gpol to replication complexes during MHV infection. (A) Cloning of MHV Pol. The pol region of gene 1 was cloned by RT-PCR. Primer-generated substitutions were made to nucleotides in both the slippery sequence (boldfaced) and the pseudoknot in order to eliminate the potential for a −1 ribosomal frameshift while maintaining the coding sequence of pol . Mutated residues are shown in red, and the resulting amino acid sequences for both wild-type Pol and cloned Pol are given above nucleotide codons. pol cDNA was subcloned into pEGFP-C1 and expressed as a fusion to the carboxy terminus of GFP (Gpol). (B and C) DBT cells on glass coverslips were transfected with cDNA encoding either GFP or Gpol. Twenty-four hours posttransfection, cells were either infected with MHV or mock infected for 5.5 h, fixed, and either imaged for GFP fluorescence (B) or subjected to indirect immunofluorescence as described in Materials and Methods by using antibodies against the MHV structural proteins M and N (C). Multinucleated cells are a cytopathic effect of viral infection. In all images, GFP is shown as green and antibody staining is shown as red. Merged images are shown with areas of colocalization in yellow.

Techniques Used: Infection, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Generated, Sequencing, Transfection, Fluorescence, Immunofluorescence, Staining

15) Product Images from ""

Article Title:

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E06-03-0249

Fibrillarin interacts with UBF in the form of a snoRNP. (A) Schematic representation of N-terminal GFP-fibrillarin fusion proteins with various truncation mutations at indicated amino acid residues. (B) Immunoprecipitations using anti-GFP antibodies in HeLa cells that were transiently transfected with a variety of constructs: beads alone (first lane, −), pEGFP-C1-Fib (second lane, FL), pEGFP-C1-Fib1 (third lane, 1), pEGFP-C1-Fib2 (fourth lane, 2), pEGFP-C1-Fib3 (fifth lane, 3), pEGFP-C1-Fib3.5 (sixth lane, 3.5), pEGFP-C1-Fib4 (seventh lane, 4), pEGFP-C1-Fib5 (eighth lane, 5), pEGFP-C1-Fib6 (ninth lane, 6), pEGFP-C1 (tenth lane, G). No plasmids were transfected into cells for lysate that was loaded into lane 11, nor was this lysate used for an immunoprecipitation. Anti-UBF (top panel), anti-Nop58 (second panel), anti-GFP (third panel), and anti-Imp4 (bottom panel) antibodies were used for detection on Western blot.
Figure Legend Snippet: Fibrillarin interacts with UBF in the form of a snoRNP. (A) Schematic representation of N-terminal GFP-fibrillarin fusion proteins with various truncation mutations at indicated amino acid residues. (B) Immunoprecipitations using anti-GFP antibodies in HeLa cells that were transiently transfected with a variety of constructs: beads alone (first lane, −), pEGFP-C1-Fib (second lane, FL), pEGFP-C1-Fib1 (third lane, 1), pEGFP-C1-Fib2 (fourth lane, 2), pEGFP-C1-Fib3 (fifth lane, 3), pEGFP-C1-Fib3.5 (sixth lane, 3.5), pEGFP-C1-Fib4 (seventh lane, 4), pEGFP-C1-Fib5 (eighth lane, 5), pEGFP-C1-Fib6 (ninth lane, 6), pEGFP-C1 (tenth lane, G). No plasmids were transfected into cells for lysate that was loaded into lane 11, nor was this lysate used for an immunoprecipitation. Anti-UBF (top panel), anti-Nop58 (second panel), anti-GFP (third panel), and anti-Imp4 (bottom panel) antibodies were used for detection on Western blot.

Techniques Used: Transfection, Construct, Immunoprecipitation, Western Blot

16) Product Images from "Recruitment of the RNA Helicase RHAU to Stress Granules via a Unique RNA-binding Domain *Recruitment of the RNA Helicase RHAU to Stress Granules via a Unique RNA-binding Domain * S⃞"

Article Title: Recruitment of the RNA Helicase RHAU to Stress Granules via a Unique RNA-binding Domain *Recruitment of the RNA Helicase RHAU to Stress Granules via a Unique RNA-binding Domain * S⃞

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M804857200

The N-terminal domain of RHAU localizes in SGs. A , scheme of wild-type RHAU and its fragments. Nter, 1-200 aa; HCR, 200-650 aa; Cter, 200-1008 aa. B , intracellular localization of different parts of RHAU. HeLa cells were transfected with an empty pEGFP-C1
Figure Legend Snippet: The N-terminal domain of RHAU localizes in SGs. A , scheme of wild-type RHAU and its fragments. Nter, 1-200 aa; HCR, 200-650 aa; Cter, 200-1008 aa. B , intracellular localization of different parts of RHAU. HeLa cells were transfected with an empty pEGFP-C1

Techniques Used: Transfection

17) Product Images from "PIN-G - A novel reporter for imaging and defining the effects of trafficking signals in membrane proteins"

Article Title: PIN-G - A novel reporter for imaging and defining the effects of trafficking signals in membrane proteins

Journal: BMC Biotechnology

doi: 10.1186/1472-6750-6-15

Cloning scheme for the preparation of the pIN-G construct and the transmembrane topology of the pIN-G reporter protein. 1A . A fragment encoding EGFP, obtained by AgeI/SalI digestion of pEGFP-C1, was ligated into an XmaI/SalI digested pDisplay backbone and the entire protein coding region PCR-amplified and ligated into a host backbone generated through NheI/KpnI digestion of pEGFP-C1 to retain the 3' multiple cloning site but exclude the EGFP coding region. The final pIN-G construct, with the appropriate reading frame throughout, was generated via a subsequent round of PCR. See Methods for further details. 1B . Following expression and cleavage of the Igk- chain leader sequence, the pIN-G protein is proposed to adopt a transmembrane orientation where HA and cMyc epitope tags and GFP have an extracellular disposition at the cell surface (or intra-lumenal in organelles or vesicular trafficking intermediates).
Figure Legend Snippet: Cloning scheme for the preparation of the pIN-G construct and the transmembrane topology of the pIN-G reporter protein. 1A . A fragment encoding EGFP, obtained by AgeI/SalI digestion of pEGFP-C1, was ligated into an XmaI/SalI digested pDisplay backbone and the entire protein coding region PCR-amplified and ligated into a host backbone generated through NheI/KpnI digestion of pEGFP-C1 to retain the 3' multiple cloning site but exclude the EGFP coding region. The final pIN-G construct, with the appropriate reading frame throughout, was generated via a subsequent round of PCR. See Methods for further details. 1B . Following expression and cleavage of the Igk- chain leader sequence, the pIN-G protein is proposed to adopt a transmembrane orientation where HA and cMyc epitope tags and GFP have an extracellular disposition at the cell surface (or intra-lumenal in organelles or vesicular trafficking intermediates).

Techniques Used: Clone Assay, Construct, Polymerase Chain Reaction, Amplification, Generated, Expressing, Sequencing

18) Product Images from "Epstein-Barr Virus BALF3 Has Nuclease Activity and Mediates Mature Virion Production during the Lytic Cycle"

Article Title: Epstein-Barr Virus BALF3 Has Nuclease Activity and Mediates Mature Virion Production during the Lytic Cycle

Journal: Journal of Virology

doi: 10.1128/JVI.00063-14

Subcellular localization of EBV BALF3 in EBV-positive cells during the lytic cycle. TW01 and NA cells were transiently transfected with pEGFP-C1-BALF3, followed by induction with 40 ng/ml TPA and 3 mM SB for 36 h, and the images shown were obtained by indirect immunofluorescence staining and photographed using a fluorescence microscope (A) and a confocal microscope (B). The detection of EBV BMRF1 with the specific antibody indicates lytic cycle activation, and the nuclei of the cells were stained with Hoechst 33258. (C) Protein extracts of cells expressing GFP-BALF3 were separated into input (I), cytosolic (C), and nuclear (N) fractions and subjected to Western blotting. After electrophoresis, GFP-BALF3 was detected by an antibody specific to GFP, and PARP1 and α-tubulin were used as markers for the nucleus and the cytoplasm, respectively.
Figure Legend Snippet: Subcellular localization of EBV BALF3 in EBV-positive cells during the lytic cycle. TW01 and NA cells were transiently transfected with pEGFP-C1-BALF3, followed by induction with 40 ng/ml TPA and 3 mM SB for 36 h, and the images shown were obtained by indirect immunofluorescence staining and photographed using a fluorescence microscope (A) and a confocal microscope (B). The detection of EBV BMRF1 with the specific antibody indicates lytic cycle activation, and the nuclei of the cells were stained with Hoechst 33258. (C) Protein extracts of cells expressing GFP-BALF3 were separated into input (I), cytosolic (C), and nuclear (N) fractions and subjected to Western blotting. After electrophoresis, GFP-BALF3 was detected by an antibody specific to GFP, and PARP1 and α-tubulin were used as markers for the nucleus and the cytoplasm, respectively.

Techniques Used: Transfection, Immunofluorescence, Staining, Fluorescence, Microscopy, Activation Assay, Expressing, Western Blot, Electrophoresis

19) Product Images from "Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation"

Article Title: Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation

Journal: Journal of Virology

doi: 10.1128/JVI.02384-16

Dependence of the subcellular localization of pUL51 and pUL89 on the presence of the other terminase subunits. (A) RPE-1 cells adenofected with the indicated BAC genomes were probed 4 days later with antibodies directed against the HA tag (for pUL51), pUL56, or pUL89 and were analyzed by confocal laser scanning microscopy. (B) HeLa cells were transfected with expression plasmids encoding pUL51, pUL56, or pUL89, either alone or in the given combinations, together with pEGFP-C1 to mark the transfected cells. After 2 days, cells were analyzed as described above for panel A. In panels A and B, contours of the nuclei are marked by white dashed lines. The numbers below each panel represent the proportion of cells exhibiting the localization pattern shown in the respective micrograph (e.g., 19 cells exhibiting the localization pattern/20 total cells). Images displaying the same antibody staining were taken with identical microscope settings. Bars, 10 μm.
Figure Legend Snippet: Dependence of the subcellular localization of pUL51 and pUL89 on the presence of the other terminase subunits. (A) RPE-1 cells adenofected with the indicated BAC genomes were probed 4 days later with antibodies directed against the HA tag (for pUL51), pUL56, or pUL89 and were analyzed by confocal laser scanning microscopy. (B) HeLa cells were transfected with expression plasmids encoding pUL51, pUL56, or pUL89, either alone or in the given combinations, together with pEGFP-C1 to mark the transfected cells. After 2 days, cells were analyzed as described above for panel A. In panels A and B, contours of the nuclei are marked by white dashed lines. The numbers below each panel represent the proportion of cells exhibiting the localization pattern shown in the respective micrograph (e.g., 19 cells exhibiting the localization pattern/20 total cells). Images displaying the same antibody staining were taken with identical microscope settings. Bars, 10 μm.

Techniques Used: BAC Assay, Confocal Laser Scanning Microscopy, Transfection, Expressing, Staining, Microscopy

20) Product Images from "Casein kinase 2 (CK2) increases survivin expression via enhanced ?-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription"

Article Title: Casein kinase 2 (CK2) increases survivin expression via enhanced ?-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription

Journal:

doi: 10.1073/pnas.0606845103

Ectopic GFP–survivin, but not GFP–CK2α, precludes TBB-induced apoptosis in HEK-293T cells. HEK-293T cells transfected with 2 μg of the plasmids pEGFP–C1, pEGFP–CK2α, or pEGFP–survivin were
Figure Legend Snippet: Ectopic GFP–survivin, but not GFP–CK2α, precludes TBB-induced apoptosis in HEK-293T cells. HEK-293T cells transfected with 2 μg of the plasmids pEGFP–C1, pEGFP–CK2α, or pEGFP–survivin were

Techniques Used: Transfection

21) Product Images from "Site-specific gene transfer with high efficiency onto a carbon nanotube-loaded electrode"

Article Title: Site-specific gene transfer with high efficiency onto a carbon nanotube-loaded electrode

Journal:

doi: 10.1098/rsif.2007.1295

Tapping mode AFM images of different kinds of electrode surfaces. ( a ) COOH-SAM surface, ( b ) CNT/PEI/COOH-SAM surface and ( c ) pEGFP-C1-adsorbed CNT/PEI/COOH-SAM surface. Scale bar, 800 nm.
Figure Legend Snippet: Tapping mode AFM images of different kinds of electrode surfaces. ( a ) COOH-SAM surface, ( b ) CNT/PEI/COOH-SAM surface and ( c ) pEGFP-C1-adsorbed CNT/PEI/COOH-SAM surface. Scale bar, 800 nm.

Techniques Used:

FACS analyses of HEK293 cells transfected with pEGFP-C1 on electrodes. ( a ) Histogram of FACS of live cells transfected on a, CNT/PEI/COOH-SAM; b, PEI/COOH-SAM electrodes; c, native cells. ( b ) Bar chart of GFP expression efficiency. The electrodes were
Figure Legend Snippet: FACS analyses of HEK293 cells transfected with pEGFP-C1 on electrodes. ( a ) Histogram of FACS of live cells transfected on a, CNT/PEI/COOH-SAM; b, PEI/COOH-SAM electrodes; c, native cells. ( b ) Bar chart of GFP expression efficiency. The electrodes were

Techniques Used: FACS, Transfection, Expressing

Fluorescent microscopic images of HEK293 cells transfected with pEGFP-C1 or pDsRed2-C1 on a CNT-loaded electrode in an arrayed fashion. Scale bar, 500 μm.
Figure Legend Snippet: Fluorescent microscopic images of HEK293 cells transfected with pEGFP-C1 or pDsRed2-C1 on a CNT-loaded electrode in an arrayed fashion. Scale bar, 500 μm.

Techniques Used: Transfection

Phase contrast and fluorescent microscopic images of HEK293 cells adhered on pEGFP-C1-adsorbed electrodes. ( a ) CNT/PEI/COOH-SAM and PEI/COOH-SAM electrodes. Plasmid DNA was adsorbed on a CNT/PEI/COOH-SAM electrode at its different concentrations. (i)
Figure Legend Snippet: Phase contrast and fluorescent microscopic images of HEK293 cells adhered on pEGFP-C1-adsorbed electrodes. ( a ) CNT/PEI/COOH-SAM and PEI/COOH-SAM electrodes. Plasmid DNA was adsorbed on a CNT/PEI/COOH-SAM electrode at its different concentrations. (i)

Techniques Used: Plasmid Preparation

( a ) FTIR-RAS and ( b ) XPS analyses of electrode surfaces. a, pEGFP-C1-adsorbed CNT/PEI/COOH-SAM electrode; b, f, CNT/PEI/COOH-SAM electrode; c, g, PEI/COOH-SAM surface; d, h, CNT/COOH-SAM electrode; e, i, COOH-SAM electrode. pEGFP-C1, FTIR spectrum of
Figure Legend Snippet: ( a ) FTIR-RAS and ( b ) XPS analyses of electrode surfaces. a, pEGFP-C1-adsorbed CNT/PEI/COOH-SAM electrode; b, f, CNT/PEI/COOH-SAM electrode; c, g, PEI/COOH-SAM surface; d, h, CNT/COOH-SAM electrode; e, i, COOH-SAM electrode. pEGFP-C1, FTIR spectrum of

Techniques Used:

22) Product Images from "BVDV Npro protein mediates the BVDV induced immunosuppression through interaction with cellular S100A9 protein"

Article Title: BVDV Npro protein mediates the BVDV induced immunosuppression through interaction with cellular S100A9 protein

Journal: Microbial Pathogenesis

doi: 10.1016/j.micpath.2018.05.047

Interaction of BVDV2a 1373 Npro and S100A9 cellular protein . The interaction of BVDV Npro with S100A9 protein in cells were confirmed by co immunoprecipitation assays. The 293T cells were co-transfected with pEGFP-C1-Npro and p3XFLAG -S100A9 plasmid or pEGFP-C1-Npro and p3XFLAG. At 24 h post transfection, cells were lysed and the proteins were isolated (by co-immunoprecipitation) using the anti-FLAG beads. Isolated proteins were analyzed for presence of GFP (e.g GFP-Npro) as well as FLAG using western blot using anti-GPF or anti-FLAG antibodies. The mock transfected 293T cells were used as negative control while cells transfected with pEGFP-C1-Npro plasmid were used as GFP-Npro positive control. Lane (1) The 293T cells without co-transfection did not show the presence of GFP-Npro or FLAG- S100A9, Lane (2) cells transfected with pEGFP-C1-Npro alone and showed the presence of GFP-Npro, Lane (3) cells transfected with p3XFLAG -S100A9 and showed the presence of S100A9, Lane (4) cells were co transfected with empty p3XFLAG along with pEGFP C1-Npro and did not show the presence of Npro (the FLAG size was 8 aa and was seen at very lower side of the gel). Lane (5) Cell lysates from cells co-transfected with p3XFLAG S100A9 along with pEGFP C1-Npro which showed the presence of S100A9 as well as Npro.
Figure Legend Snippet: Interaction of BVDV2a 1373 Npro and S100A9 cellular protein . The interaction of BVDV Npro with S100A9 protein in cells were confirmed by co immunoprecipitation assays. The 293T cells were co-transfected with pEGFP-C1-Npro and p3XFLAG -S100A9 plasmid or pEGFP-C1-Npro and p3XFLAG. At 24 h post transfection, cells were lysed and the proteins were isolated (by co-immunoprecipitation) using the anti-FLAG beads. Isolated proteins were analyzed for presence of GFP (e.g GFP-Npro) as well as FLAG using western blot using anti-GPF or anti-FLAG antibodies. The mock transfected 293T cells were used as negative control while cells transfected with pEGFP-C1-Npro plasmid were used as GFP-Npro positive control. Lane (1) The 293T cells without co-transfection did not show the presence of GFP-Npro or FLAG- S100A9, Lane (2) cells transfected with pEGFP-C1-Npro alone and showed the presence of GFP-Npro, Lane (3) cells transfected with p3XFLAG -S100A9 and showed the presence of S100A9, Lane (4) cells were co transfected with empty p3XFLAG along with pEGFP C1-Npro and did not show the presence of Npro (the FLAG size was 8 aa and was seen at very lower side of the gel). Lane (5) Cell lysates from cells co-transfected with p3XFLAG S100A9 along with pEGFP C1-Npro which showed the presence of S100A9 as well as Npro.

Techniques Used: Immunoprecipitation, Transfection, Plasmid Preparation, Isolation, Western Blot, Negative Control, Positive Control, Cotransfection

23) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

24) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

25) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

26) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

27) Product Images from "Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids"

Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

Journal: PLoS ONE

doi: 10.1371/journal.pone.0043283

Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
Figure Legend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

Techniques Used: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.
Figure Legend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

Techniques Used: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

28) Product Images from "Immunization with a DNA vaccine encoding Toxoplasma gondii Superoxide dismutase (TgSOD) induces partial immune protection against acute toxoplasmosis in BALB/c mice"

Article Title: Immunization with a DNA vaccine encoding Toxoplasma gondii Superoxide dismutase (TgSOD) induces partial immune protection against acute toxoplasmosis in BALB/c mice

Journal: BMC Infectious Diseases

doi: 10.1186/s12879-017-2507-5

Identification of TgSOD expression in vitro by fluorescence microscopic detection and Western blotting. Fluorescence microscopy images of TgSOD protein in ( a ) HEK 293 T cells that were transfected with pEGFP-SOD and ( b ) empty plasmid pEGFP-C1, and ( c ) non-transfected HEK 293 T cells; ( d ) Western blotting of pEGFP-SOD expressed in HEK 293 T cells (lane 1) probed with anti-STAg mouse sera as primary antibody and the protein of SOD is 23 KDa, whereas no band in the negative control cells with the empty plasmid pEGFP-C1 (lane 2) and GAPDH serves as a loading control
Figure Legend Snippet: Identification of TgSOD expression in vitro by fluorescence microscopic detection and Western blotting. Fluorescence microscopy images of TgSOD protein in ( a ) HEK 293 T cells that were transfected with pEGFP-SOD and ( b ) empty plasmid pEGFP-C1, and ( c ) non-transfected HEK 293 T cells; ( d ) Western blotting of pEGFP-SOD expressed in HEK 293 T cells (lane 1) probed with anti-STAg mouse sera as primary antibody and the protein of SOD is 23 KDa, whereas no band in the negative control cells with the empty plasmid pEGFP-C1 (lane 2) and GAPDH serves as a loading control

Techniques Used: Expressing, In Vitro, Fluorescence, Western Blot, Microscopy, Transfection, Plasmid Preparation, Negative Control

Toxoplasma -specific antibody levels in the sera of immunized BALB/c mice. The total IgG antibodies ( a ) in the collected serum samples of BALB/c mice immunized with pEGFP-SOD, pEGFP-C1, PBS and blank control on weeks 0, 2, 4, 8 were analyzed by ELISA. The levels of IgG1 and IgG2a ( b ) subtypes in the sera 28 days after the last immunization were determined by ELISA. The results are expressed as the means ± SD from three independent experiments . p
Figure Legend Snippet: Toxoplasma -specific antibody levels in the sera of immunized BALB/c mice. The total IgG antibodies ( a ) in the collected serum samples of BALB/c mice immunized with pEGFP-SOD, pEGFP-C1, PBS and blank control on weeks 0, 2, 4, 8 were analyzed by ELISA. The levels of IgG1 and IgG2a ( b ) subtypes in the sera 28 days after the last immunization were determined by ELISA. The results are expressed as the means ± SD from three independent experiments . p

Techniques Used: Mouse Assay, Enzyme-linked Immunosorbent Assay

29) Product Images from "Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells"

Article Title: Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.22.7807-7816.2001

Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.
Figure Legend Snippet: Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.

Techniques Used: Northern Blot, Transfection, Isolation, Plasmid Preparation, Labeling, Sequencing

dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.

Techniques Used: Produced, Sequencing, Inhibition, Expressing, In Situ, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, In Vitro, Staining

Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.
Figure Legend Snippet: Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.

Techniques Used: Sequencing, Activity Assay, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation

Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.
Figure Legend Snippet: Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.

Techniques Used: Activity Assay, Sequencing, Inhibition, Plasmid Preparation, Transfection, Fluorescence, Standard Deviation, Cell Culture

Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.

Techniques Used: Sequencing, Inhibition, Expressing, In Vitro, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, Microscopy, Staining

30) Product Images from "Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro"

Article Title: Translation of Polioviral mRNA Is Inhibited by Cleavage of Polypyrimidine Tract-Binding Proteins Executed by Polioviral 3Cpro

Journal: Journal of Virology

doi:

Effect of PTB and its derivatives on poliovirus IRES activity. (A) Schematic diagram of the reporter plasmid. This plasmid expresses a dicistronic mRNA consisting of the Renilla luciferase (RLuc) gene at the first cistron and the polioviral IRES-firefly luciferase (FLuc) gene at the second cistron. The RLuc and FLuc are directed by scanning and IRES-dependent translation, respectively. (B) Schematic diagram of effector plasmids. These plasmids produce full-length PTB4 or PTB1 and PTB4 derivatives fused with GFP. (C) Effect of PTB or its derivatives on poliovirus IRES activity. 293T cells were cotransfected with 0.75 μg of dicistronic plasmid and effector plasmids as indicated in the chart at the bottom. The total amount of effector plasmids was maintained at a constant level by adding control plasmid pEGFP-C1 when necessary. Forty-eight hours after transfection, Renilla luciferase and firefly luciferase activities were measured as described in Materials and Methods, and the relative ratio of firefly luciferase to Renilla luciferase activity in each cell lysate was calculated. The columns and bars represent the means and standard deviations of four independent transfection experiments. The numbers in the chart represent micrograms of DNA.
Figure Legend Snippet: Effect of PTB and its derivatives on poliovirus IRES activity. (A) Schematic diagram of the reporter plasmid. This plasmid expresses a dicistronic mRNA consisting of the Renilla luciferase (RLuc) gene at the first cistron and the polioviral IRES-firefly luciferase (FLuc) gene at the second cistron. The RLuc and FLuc are directed by scanning and IRES-dependent translation, respectively. (B) Schematic diagram of effector plasmids. These plasmids produce full-length PTB4 or PTB1 and PTB4 derivatives fused with GFP. (C) Effect of PTB or its derivatives on poliovirus IRES activity. 293T cells were cotransfected with 0.75 μg of dicistronic plasmid and effector plasmids as indicated in the chart at the bottom. The total amount of effector plasmids was maintained at a constant level by adding control plasmid pEGFP-C1 when necessary. Forty-eight hours after transfection, Renilla luciferase and firefly luciferase activities were measured as described in Materials and Methods, and the relative ratio of firefly luciferase to Renilla luciferase activity in each cell lysate was calculated. The columns and bars represent the means and standard deviations of four independent transfection experiments. The numbers in the chart represent micrograms of DNA.

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

31) Product Images from "Nucleocytoplasmic Shuttling of Pak5 Regulates Its Antiapoptotic Properties †"

Article Title: Nucleocytoplasmic Shuttling of Pak5 Regulates Its Antiapoptotic Properties †

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.26.8.3215-3230.2006

Mapping of a minimal region in Pak5 for localization to mitochondria. (A) Schematic representation of different Pak5 deletion constructs. (B) Confocal microscopy. CHO cells were transfected with pEGFP-C1 vector or the indicated constructs. Cells were stained with MitoTracker and fixed 24 h after transfection, as described in Materials and Methods. The fields shown were analyzed independently by confocal fluorescence microscopy at the appropriate wavelengths for GFP (Pak5) and MitoTracker red CMXRos (Mito), and the two images were overlaid (overlay). Bar = 20 μm. In parallel with microscopy, cellular fractionation was performed for each construct 48 h after transfections, as described in Materials and Methods, to assess the presence of Pak5 in the cellular compartments. Equal amounts of proteins from the cytosolic (S100), light microsome (L), and mitochondrial (M) fractions were loaded onto a gel, and immunoblotting was performed using an anti-GFP antibody. Anti-COX IV (for mitochondria) and anticalnexin (for light membranes) antibodies were used as controls. (C) CHO cells were transfected with empty vector (pCMV6) or with a vector carrying Myc-wt-Pak5 or Myc-mut-Pak5 cDNA. Nuclear extraction was performed as described in Materials and Methods. Equal amounts of lysates were loaded onto a gel and subjected to immunoblot analysis using anti-Myc antibodies. (D) CHO cells were transiently transfected with pCMV6 Myc-wt-Pak5 or pCMV6 Myc-mut-Pak5. Cells were processed for immunofluorescence using MitoTracker anti-Myc antibodies as described in Materials and Methods. Bar = 20 μm. (E) CHO cells were transfected with a GFP vector encoding GFP fused to the mitochondrial targeting sequence, corresponding to amino acids 30 to 83 of the Pak5 sequence. For the first three panels, cells were stained with MitoTracker red CMXRos (Mito) and then fixed in methanol 24 h after transfection, and confocal microscopy was used to visualize colocalization. For the right panel, cellular fractionation was performed to assess the presence of Pak5 in the different cellular fractions. Equal amounts of proteins from the cytosolic S100, light microsome (L), and mitochondrial (M) fractions were loaded on a gel, and immunoblotting was performed using anti-GFP antibodies. Anti-COX IV (for mitochondria) and anticalnexin (for light membranes) antibodies were used as controls. Bar = 20 μm.
Figure Legend Snippet: Mapping of a minimal region in Pak5 for localization to mitochondria. (A) Schematic representation of different Pak5 deletion constructs. (B) Confocal microscopy. CHO cells were transfected with pEGFP-C1 vector or the indicated constructs. Cells were stained with MitoTracker and fixed 24 h after transfection, as described in Materials and Methods. The fields shown were analyzed independently by confocal fluorescence microscopy at the appropriate wavelengths for GFP (Pak5) and MitoTracker red CMXRos (Mito), and the two images were overlaid (overlay). Bar = 20 μm. In parallel with microscopy, cellular fractionation was performed for each construct 48 h after transfections, as described in Materials and Methods, to assess the presence of Pak5 in the cellular compartments. Equal amounts of proteins from the cytosolic (S100), light microsome (L), and mitochondrial (M) fractions were loaded onto a gel, and immunoblotting was performed using an anti-GFP antibody. Anti-COX IV (for mitochondria) and anticalnexin (for light membranes) antibodies were used as controls. (C) CHO cells were transfected with empty vector (pCMV6) or with a vector carrying Myc-wt-Pak5 or Myc-mut-Pak5 cDNA. Nuclear extraction was performed as described in Materials and Methods. Equal amounts of lysates were loaded onto a gel and subjected to immunoblot analysis using anti-Myc antibodies. (D) CHO cells were transiently transfected with pCMV6 Myc-wt-Pak5 or pCMV6 Myc-mut-Pak5. Cells were processed for immunofluorescence using MitoTracker anti-Myc antibodies as described in Materials and Methods. Bar = 20 μm. (E) CHO cells were transfected with a GFP vector encoding GFP fused to the mitochondrial targeting sequence, corresponding to amino acids 30 to 83 of the Pak5 sequence. For the first three panels, cells were stained with MitoTracker red CMXRos (Mito) and then fixed in methanol 24 h after transfection, and confocal microscopy was used to visualize colocalization. For the right panel, cellular fractionation was performed to assess the presence of Pak5 in the different cellular fractions. Equal amounts of proteins from the cytosolic S100, light microsome (L), and mitochondrial (M) fractions were loaded on a gel, and immunoblotting was performed using anti-GFP antibodies. Anti-COX IV (for mitochondria) and anticalnexin (for light membranes) antibodies were used as controls. Bar = 20 μm.

Techniques Used: Construct, Confocal Microscopy, Transfection, Plasmid Preparation, Staining, Fluorescence, Microscopy, Cell Fractionation, Immunofluorescence, Sequencing

32) Product Images from "Benzyl butyl phthalate induces migration, invasion, and angiogenesis of Huh7 hepatocellular carcinoma cells through nongenomic AhR/G-protein signaling"

Article Title: Benzyl butyl phthalate induces migration, invasion, and angiogenesis of Huh7 hepatocellular carcinoma cells through nongenomic AhR/G-protein signaling

Journal: BMC Cancer

doi: 10.1186/1471-2407-14-556

BBP activates AhR at the cell membrane, which interacts with G proteins. (A) Huh7 cells were transfected with pEGFP-C1-AhR or pEGFP-C1 as a plasmid control. Cells were stimulated by adding DMSO or BBP (1 μM) and then analyzed by real-time TIRF microscopy. Scale bars: 10 μm. The left panel shows Huh7 cells transfected with the pEGFP-C1 plasmid control treated with DMSO (upper) or BBP (lower). The middle panel shows Huh7 cells transfected with pEGFP-C1-AhR and then treated with DMSO (upper) or BBP (lower). The increased intensity of GFP fluorescence indicates AhR expression at the cell membrane, which was induced by BBP. The right panel shows the GFP intensity analyzed by Axio Vision Rel 4.8 software. (B) Huh7 cells were transfected with pEGFP-C1-AhR and then stimulated with BBP (1 μM) before analyzed by real-time confocal microscopy (upper panel), Scale bars: 10 μm. The GFP intensity was analyzed by FV10-ASW 2.1 software (Olympus) (lower panel). (C) Expression of Gα q/11 and G β proteins after BBP treatment for the indicated time was detected by immunoblotting. β-actin was used as an internal control. (D) Interaction of AhR with Gα q/11 at the cell membrane after treatment with BBP (1 μM) was imaged by double immunogold electron microscopy. Black arrows indicate Gα q/11 , and the white arrows indicate AhR. The localization of G protein and AhR protein are shown. (upper left panel) shows the untreatment group and (Lower left, Right left panel) indicated BBP treatment groups. Scale bars: 500 nm. CM, cell membrane; N, nucleus. (E) Huh7 cells after 30 minutes of treatment with BBP (1 μM) or DMSO as the control. The interaction of AhR with Gα q/11 and G β was detected by immunoprecipitation (IP) followed by immunoblot analysis. The IgG were used cell lysates mixed with control and BBP treatment group. Normal rabbit IgG were used as negative control. (F) Huh7 cells were transfected with two different AhR shRNAs as described the Methods or a control shRNA. After treatment with or without BBP (1 μM), AhR, Gα q/11 , and G β levels were measured by immunoblotting. β-actin was used as an internal control.
Figure Legend Snippet: BBP activates AhR at the cell membrane, which interacts with G proteins. (A) Huh7 cells were transfected with pEGFP-C1-AhR or pEGFP-C1 as a plasmid control. Cells were stimulated by adding DMSO or BBP (1 μM) and then analyzed by real-time TIRF microscopy. Scale bars: 10 μm. The left panel shows Huh7 cells transfected with the pEGFP-C1 plasmid control treated with DMSO (upper) or BBP (lower). The middle panel shows Huh7 cells transfected with pEGFP-C1-AhR and then treated with DMSO (upper) or BBP (lower). The increased intensity of GFP fluorescence indicates AhR expression at the cell membrane, which was induced by BBP. The right panel shows the GFP intensity analyzed by Axio Vision Rel 4.8 software. (B) Huh7 cells were transfected with pEGFP-C1-AhR and then stimulated with BBP (1 μM) before analyzed by real-time confocal microscopy (upper panel), Scale bars: 10 μm. The GFP intensity was analyzed by FV10-ASW 2.1 software (Olympus) (lower panel). (C) Expression of Gα q/11 and G β proteins after BBP treatment for the indicated time was detected by immunoblotting. β-actin was used as an internal control. (D) Interaction of AhR with Gα q/11 at the cell membrane after treatment with BBP (1 μM) was imaged by double immunogold electron microscopy. Black arrows indicate Gα q/11 , and the white arrows indicate AhR. The localization of G protein and AhR protein are shown. (upper left panel) shows the untreatment group and (Lower left, Right left panel) indicated BBP treatment groups. Scale bars: 500 nm. CM, cell membrane; N, nucleus. (E) Huh7 cells after 30 minutes of treatment with BBP (1 μM) or DMSO as the control. The interaction of AhR with Gα q/11 and G β was detected by immunoprecipitation (IP) followed by immunoblot analysis. The IgG were used cell lysates mixed with control and BBP treatment group. Normal rabbit IgG were used as negative control. (F) Huh7 cells were transfected with two different AhR shRNAs as described the Methods or a control shRNA. After treatment with or without BBP (1 μM), AhR, Gα q/11 , and G β levels were measured by immunoblotting. β-actin was used as an internal control.

Techniques Used: Transfection, Plasmid Preparation, Microscopy, Fluorescence, Expressing, Software, Confocal Microscopy, Electron Microscopy, Immunoprecipitation, Negative Control, shRNA

33) Product Images from "Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus"

Article Title: Role of TAF4 in Transcriptional Activation by Rta of Epstein-Barr Virus

Journal: PLoS ONE

doi: 10.1371/journal.pone.0054075

Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).
Figure Legend Snippet: Mapping the interaction domains in TAF4 and Rta. (A) Plasmids that expressed deleted GFP-TAF4 were used to delineate the region in TAF4 that interacts with Rta. Numbers represent the positions of amino acids in TAF4. Q denotes the glutamine-rich regions. (B) 293T cells were cotransfected with pCMV-R and plasmids that expressed GFP fusion proteins including pEGFP-TAF4 (lanes 2, 7), pEGFP-TAF4-NM (lanes 3, 8), pEGFP-TAF4-C (lanes 4 and 9) or pEGFP-C1 (lanes 1, 6). Input lanes were loaded with 5% of the lysate (lanes 1–5). Proteins in the lysates were coimmunoprecipitated (IP) with anti-GFP antibody and analyzed by immunoblotting (IB) using anti-Rta antibody (lanes 6–9). (C) Deletion mutants of Rta were used to identify the region in Rta that interacts with TAF4. Numbers represent the positions of amino acids in Rta (D). Plasmids that expressed GFP-Rta (lanes 2, 7), GFP-N190 (lanes 3, 8), GFP-N191-415 (lanes 4, 9), GFP-Rev (lanes 5, 10) or GFP (lanes 1, 6) were transfected into 293T cells. The input lanes were loaded with 5% of the cell lysates and GFP-fusion proteins were detected using anti-GFP antibody (lanes 1–5). Proteins in the lysates were coimmunoprecipitated with anti-TAF4 antibody and analyzed by immunoblotting using anti-GFP antibody (lanes 6–10).

Techniques Used: Transfection

Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.
Figure Legend Snippet: Indirect immunofluorescence analysis. P3HR1 cells were transfected with pEGFP-C1 (A–D) or pEGFP-TAF4 (E–H) and then treated with sodium butyrate for 24 hr. Cells were incubated with monoclonal anti-Rta antibody and observed under a confocal laser-scanning microscope. DAPI staining revealed the positions of nuclei (A and E). D and H are merged images.

Techniques Used: Immunofluorescence, Transfection, Incubation, Laser-Scanning Microscopy, Staining

34) Product Images from "Interleukin-17 augments tumor necrosis factor α-mediated increase of hypoxia-inducible factor-1α and inhibits vasodilator-stimulated phosphoprotein expression to reduce the adhesion of breast cancer cells"

Article Title: Interleukin-17 augments tumor necrosis factor α-mediated increase of hypoxia-inducible factor-1α and inhibits vasodilator-stimulated phosphoprotein expression to reduce the adhesion of breast cancer cells

Journal: Oncology Letters

doi: 10.3892/ol.2017.5825

Transfection with HIF-1α-siRNA mitigated the reduction of VASP expression and promoted adhesive ability in the MDA-MB-231 cells treated with IL-17 and TNF-α. (A) MDA-MB-231 cells were transfected with siRNA-HIF-1α or pEGFP-C1-HIF-1α for 24 h. Scrambled siRNA and pEGFP-C1 vector transfection was used as the controls. The HIF-1α and VASP protein expression levels were determined using western blotting. GAPDH was used as a loading control. (B) Cells were treated 100 ng/ml IL-17 or 1 ng/ml TNF-α or in combination following transfection with siRNA-HIF-1α for 24 h. Scrambled siRNA was used as a control. The HIF-1α and VASP protein expression levels were determined using western blotting. (C) Cell adhesion was examined using an MTT assay. The results were presented as the mean + standard deviation of 3 independent experiments. *P
Figure Legend Snippet: Transfection with HIF-1α-siRNA mitigated the reduction of VASP expression and promoted adhesive ability in the MDA-MB-231 cells treated with IL-17 and TNF-α. (A) MDA-MB-231 cells were transfected with siRNA-HIF-1α or pEGFP-C1-HIF-1α for 24 h. Scrambled siRNA and pEGFP-C1 vector transfection was used as the controls. The HIF-1α and VASP protein expression levels were determined using western blotting. GAPDH was used as a loading control. (B) Cells were treated 100 ng/ml IL-17 or 1 ng/ml TNF-α or in combination following transfection with siRNA-HIF-1α for 24 h. Scrambled siRNA was used as a control. The HIF-1α and VASP protein expression levels were determined using western blotting. (C) Cell adhesion was examined using an MTT assay. The results were presented as the mean + standard deviation of 3 independent experiments. *P

Techniques Used: Transfection, Expressing, Multiple Displacement Amplification, Plasmid Preparation, Western Blot, MTT Assay, Standard Deviation

35) Product Images from "Functional Cooperation of Epstein-Barr Virus Nuclear Antigen 2 and the Survival Motor Neuron Protein in Transactivation of the Viral LMP1 Promoter"

Article Title: Functional Cooperation of Epstein-Barr Virus Nuclear Antigen 2 and the Survival Motor Neuron Protein in Transactivation of the Viral LMP1 Promoter

Journal: Journal of Virology

doi: 10.1128/JVI.75.23.11781-11790.2001

(A) Interaction of DP103 and SMN in B lymphocytes. Coimmunoprecipitations (IP) from Raji cell extracts were performed with DP103-specific MAb 9A3 (IP: DP103 Ab) or an irrelevant control antibody (anti-TrypE 3A6, IP: control Ab), followed by SDS–10% PAGE and Western blotting. Precipitated proteins were detected with anti-SMN-N serum (Santa Cruz Biochemicals) (left panel, WB: anti SMN) or anti-DP103 MAb 8H4 (right panel, WB: anti DP103). The positions of SMN and DP103 are indicated by arrows. Lanes designated Raji input represent ca. 1% of unprecipitated Raji cell extract. The positions of the molecular mass marker proteins are indicated on the left side (in kilodaltons). (B) SMN coactivates the viral LMP1 promoter in the presence of EBNA2. BJAB cells were transfected with luciferase reporter constructs encoding positions −327/+40 (EBNA2 responsive) or −154/+40 (nonresponsive) of the LMP1 promoter (4 μg) and the indicated combinations of pSG5 constructs encoding EBNA2 or HA-tagged SMN and DP103 (10 μg). After 48 h, the cells were lysed by freeze-thawing, and the luciferase activity was measured. The transfection efficiency was determined by scanning the expression of cotransfected pEGFP-C1 vector (2 μg) by FACS analysis prior to lysis of the cells. For each experiment, luciferase values standardized for transfection efficiency were calculated relative to the values obtained by EBNA2 and the respective full-length promoter construct (set to 100%). Graphs represent the mean values of five independent experiments (± the standard error of the mean [SEM]).
Figure Legend Snippet: (A) Interaction of DP103 and SMN in B lymphocytes. Coimmunoprecipitations (IP) from Raji cell extracts were performed with DP103-specific MAb 9A3 (IP: DP103 Ab) or an irrelevant control antibody (anti-TrypE 3A6, IP: control Ab), followed by SDS–10% PAGE and Western blotting. Precipitated proteins were detected with anti-SMN-N serum (Santa Cruz Biochemicals) (left panel, WB: anti SMN) or anti-DP103 MAb 8H4 (right panel, WB: anti DP103). The positions of SMN and DP103 are indicated by arrows. Lanes designated Raji input represent ca. 1% of unprecipitated Raji cell extract. The positions of the molecular mass marker proteins are indicated on the left side (in kilodaltons). (B) SMN coactivates the viral LMP1 promoter in the presence of EBNA2. BJAB cells were transfected with luciferase reporter constructs encoding positions −327/+40 (EBNA2 responsive) or −154/+40 (nonresponsive) of the LMP1 promoter (4 μg) and the indicated combinations of pSG5 constructs encoding EBNA2 or HA-tagged SMN and DP103 (10 μg). After 48 h, the cells were lysed by freeze-thawing, and the luciferase activity was measured. The transfection efficiency was determined by scanning the expression of cotransfected pEGFP-C1 vector (2 μg) by FACS analysis prior to lysis of the cells. For each experiment, luciferase values standardized for transfection efficiency were calculated relative to the values obtained by EBNA2 and the respective full-length promoter construct (set to 100%). Graphs represent the mean values of five independent experiments (± the standard error of the mean [SEM]).

Techniques Used: Polyacrylamide Gel Electrophoresis, Western Blot, Marker, Transfection, Luciferase, Construct, Activity Assay, Expressing, Plasmid Preparation, FACS, Lysis

36) Product Images from "The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3"

Article Title: The Ebola Virus VP35 Protein Inhibits Activation of Interferon Regulatory Factor 3

Journal: Journal of Virology

doi: 10.1128/JVI.77.14.7945-7956.2003

The Ebola virus VP35 protein prevents the nuclear translocation of hIRF-3 after SeV infection. (A) Fluorescence images showing expression of HA-tagged VP35(R) protein (red) and the corresponding distribution of GFP-hIRF-3 (green). Cells expressing both HA-tagged VP35 and GFP-IRF-3 are indicated by the large white arrows. Examples of cells with nuclear GFP-IRF-3 are indicated by the small yellow arrows. Vero cells were transfected with 0.4 μg of VP35(R) expression plasmid plus 0.8 μg of pEGFP-C1-hIRF3 and infected 24 h later with SeV. Eight hours postinfection, cells were fixed and stained with anti-HA monoclonal antibody (red). (B) The percentage of GFP-IRF-3-expressing cells with nuclear GFP-IRF-3 in cells transfected with the indicated plasmids and either mock infected or infected with SeV is shown. Vero cells were transfected with 0.4 μg of empty vector or expression plasmids for Ebola virus Zaire VP35 [VP35(Z)], HA-tagged Ebola virus Reston VP35 [HAVP35(R)], influenza virus NS1 protein (NS1), or Ebola virus Zaire virus VP24 protein (VP24), plus 0.2 μg of pEGFP-C1-hIRF3. At 24 h posttransfection, the cells were mock infected or infected with SeV at an MOI of 10. Eight hours postinfection, the cells were examined for GFP localization. The results are the average of two independent experiments where 200 to 300 cells were counted per transfection.
Figure Legend Snippet: The Ebola virus VP35 protein prevents the nuclear translocation of hIRF-3 after SeV infection. (A) Fluorescence images showing expression of HA-tagged VP35(R) protein (red) and the corresponding distribution of GFP-hIRF-3 (green). Cells expressing both HA-tagged VP35 and GFP-IRF-3 are indicated by the large white arrows. Examples of cells with nuclear GFP-IRF-3 are indicated by the small yellow arrows. Vero cells were transfected with 0.4 μg of VP35(R) expression plasmid plus 0.8 μg of pEGFP-C1-hIRF3 and infected 24 h later with SeV. Eight hours postinfection, cells were fixed and stained with anti-HA monoclonal antibody (red). (B) The percentage of GFP-IRF-3-expressing cells with nuclear GFP-IRF-3 in cells transfected with the indicated plasmids and either mock infected or infected with SeV is shown. Vero cells were transfected with 0.4 μg of empty vector or expression plasmids for Ebola virus Zaire VP35 [VP35(Z)], HA-tagged Ebola virus Reston VP35 [HAVP35(R)], influenza virus NS1 protein (NS1), or Ebola virus Zaire virus VP24 protein (VP24), plus 0.2 μg of pEGFP-C1-hIRF3. At 24 h posttransfection, the cells were mock infected or infected with SeV at an MOI of 10. Eight hours postinfection, the cells were examined for GFP localization. The results are the average of two independent experiments where 200 to 300 cells were counted per transfection.

Techniques Used: Translocation Assay, Infection, Fluorescence, Expressing, Transfection, Plasmid Preparation, Staining

37) Product Images from "Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells"

Article Title: Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

Journal: Molecular and Cellular Biology

doi: 10.1128/MCB.21.22.7807-7816.2001

Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.
Figure Legend Snippet: Northern analysis of cognate (EGFP) and the noncognate (β-galactosidase) mRNAs. Undifferentiated ES cells were grown on the feeder layer and transfected by three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 1, 2, and 3, respectively). As a control, ES cells were transfected with three plasmids, pCMV-lacZ (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, 0, 1, or 2 μg (lanes 4, 5, and 6, respectively). Total RNA was isolated from transfected cells, and 25 μg of total RNA was loaded in each lane. The EGFP probe was a 0.7-kb fragment isolated from the pEGFP-C1 plasmid, and the lacZ probe was a 2.5-kb fragment from the pCMV-lacZ plasmid. The probes were labeled by [α- 32 P]dCTP. A cDNA probe corresponding to the mouse β-actin coding sequence was hybridized as a control.

Techniques Used: Northern Blot, Transfection, Isolation, Plasmid Preparation, Labeling, Sequencing

dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: dsRNA produced a sequence-specific and dose-dependent gene silencing in Drosophila S2 cells. (A) Inhibition of EGFP expression by in situ production of dsRNA. S2 cells were transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. Throughout all transfections, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding lacZ , pCMV-lacZ, was used instead of pEGFP-C1. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection experiments performed in duplicate. (B) Sequence-specific and dose-dependent inhibition of EGFP by the in vitro-transcribed dsRNA. S2 cells were transfected with 2.5 μg of pIZ/US9-GFP plasmid and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP (lanes 1, 2, and 3, respectively) using a calcium phosphate method. Photographs were taken 72 h later, depicted by a bright field (upper panel) and a fluorescence micrograph (lower panel). (C) β-Galactosidase expression is not inhibited by in-vitro transcribed dsRNA-EGFP. As a control, S2 cells were transfected with 2.5 μg of pActin-lacZ and 0, 1.5, or 3.0 μg of the in vitro-transcribed dsRNA-EGFP by a calcium phosphate method. Histochemical staining was carried out 72 h later.

Techniques Used: Produced, Sequencing, Inhibition, Expressing, In Situ, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, In Vitro, Staining

Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.
Figure Legend Snippet: Several mammalian cells do not show sequence-specific RNAi activity. Three mammalian cell lines, BsrT7/5 (A), STO (B), and CHO-K1 (C), were tested for RNAi activity by transient transfection of three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and increasing amounts of pGEMT-dsEGFP (0.25 to 2 μg). Throughout transfection, the total amount of DNA was held constant by addition of unrelated pUC19 plasmid. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfections of duplicate samples.

Techniques Used: Sequencing, Activity Assay, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation

Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.
Figure Legend Snippet: Undifferentiated ES cells exhibit RNAi activity. (A) Sequence-specific and dose-dependent inhibition of EGFP by pGEMT-dsEGFP plasmid in ES cells grown on a feeder layer. ES cells were plated on STO feeder cells and transfected with three plasmids, pEGFP-C1 (1 μg), pSC6-T7-Neo (1 μg), and an increasing amount of pGEMT-dsEGFP, ranging from 0.25 to 1 μg. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no pGEMT-dsEGFP plasmid. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least five separate transfection experiments performed in duplicate. (B) Non-sequence-specific inhibition of EGFP by pGEMT-dsEGFP plasmid in differentiated ES cells cultured without the feeder layer. The same experiment was carried out in ES cells plated directly on a gelatin-coated plate with no feeder cells.

Techniques Used: Activity Assay, Sequencing, Inhibition, Plasmid Preparation, Transfection, Fluorescence, Standard Deviation, Cell Culture

Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.
Figure Legend Snippet: Sequence-specific and dose-dependent inhibition of EGFP expression by in-vitro transcribed dsRNA in undifferentiated ES cells. (A) ES cells were plated on feeder cells and transfected with 1 μg of the pEGFP-C1 plasmid and increasing amounts, 0.25 to 1.0 μg, of the in vitro-transcribed dsRNA. To test the sequence specificity of RNAi, 1 μg of the plasmid encoding the β-galactosidase, pCMV-lacZ, was used as a control. The RLUs of fluorescence or chemiluminescence were normalized to that of lysate containing no dsRNA. The relative activities of cells transfected with pEGFP-C1 plasmid (solid bars) and pCMV-lacZ plasmid (open bars) are shown. Standard deviation indicates the variation among at least three separate transfection performed in duplicate. (B) Fluorescence microscopy of undifferentiated ES cells transfected with 2.5 μg of pEGFP-C1 plasmid and an increasing amount, 0, 1, and 2 μg (lanes, 1, 2, and 3, respectively), of the in vitro-transcribed dsRNA-EGFP. Photographs were taken 72 h later, using a bright field (upper panel) and fluorescence (lower panel). (C) β-Galactosidase expression is not inhibited by in vitro-transcribed dsRNA-EGFP. ES cells were transfected with 2.5 μg of pCMV-lacZ and 0, 1, or 2 μg of in vitro-transcribed dsRNA-EGFP. Histochemical staining was carried out 72 h later.

Techniques Used: Sequencing, Inhibition, Expressing, In Vitro, Transfection, Plasmid Preparation, Fluorescence, Standard Deviation, Microscopy, Staining

38) Product Images from "RNase L restricts the mobility of engineered retrotransposons in cultured human cells"

Article Title: RNase L restricts the mobility of engineered retrotransposons in cultured human cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt1308

Expression of RNase L reduces L1 protein expression. ( A ) L1 protein expression: HeLa-M cells were co-transfected with pAD2TE1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Two days after transfection, cells were selected with hygromycin containing medium for an additional 4 days when total cell lysates and L1 RNPs were prepared. Western blotting, using anti-T7 and anti-TAP antibodies, was used to detect ORF1p and ORF2p, respectively. Shown are two exposures of the ORF2p anti-TAP western blot. Endogenous ribosomal S6 protein was used as the loading/transfer control. β-Actin detection discriminated the total cell lysate (left side of panel) from the L1 RNP fractions (right side of panel). The experiments were repeated twice (biological replicates) with similar results. Shown are data from one representative experiment. ( B ) RNase L does not inhibit exogenous EGFP protein expression: HeLa-M cells were co-transfected with pEGFP-C1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Total cell lysates were harvested and the expression of RNase L and GFP was detected in western blot experiments using anti-RNase L and anti-GFP antibodies at 48 h after transfection. GAPDH served as a loading and transfer control.
Figure Legend Snippet: Expression of RNase L reduces L1 protein expression. ( A ) L1 protein expression: HeLa-M cells were co-transfected with pAD2TE1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Two days after transfection, cells were selected with hygromycin containing medium for an additional 4 days when total cell lysates and L1 RNPs were prepared. Western blotting, using anti-T7 and anti-TAP antibodies, was used to detect ORF1p and ORF2p, respectively. Shown are two exposures of the ORF2p anti-TAP western blot. Endogenous ribosomal S6 protein was used as the loading/transfer control. β-Actin detection discriminated the total cell lysate (left side of panel) from the L1 RNP fractions (right side of panel). The experiments were repeated twice (biological replicates) with similar results. Shown are data from one representative experiment. ( B ) RNase L does not inhibit exogenous EGFP protein expression: HeLa-M cells were co-transfected with pEGFP-C1 and either an empty vector (pFLAG-CMV-2) or a plasmid that encodes an amino-terminal FLAG-tagged RNase L expression plasmid. Total cell lysates were harvested and the expression of RNase L and GFP was detected in western blot experiments using anti-RNase L and anti-GFP antibodies at 48 h after transfection. GAPDH served as a loading and transfer control.

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

39) Product Images from "Serum starvation raises turnover of phosphorylated p62/SQSTM1 (Serine 349), reveals expression of proteasome and N-glycanase1 interactive protein RAD23B and sensitizes human synovial fibroblasts to BAY 11-7085-induced cell death"

Article Title: Serum starvation raises turnover of phosphorylated p62/SQSTM1 (Serine 349), reveals expression of proteasome and N-glycanase1 interactive protein RAD23B and sensitizes human synovial fibroblasts to BAY 11-7085-induced cell death

Journal: Oncotarget

doi: 10.18632/oncotarget.26295

Starvation increases ENGase turnover in synovial fibroblasts Co-transfection of NGLY1 with p62 increases p62 expression in HEK293 cells. ( A ) Synovial fibroblasts were cultured with or without serum, in the presence of different concentrations of MG132 as indicated. Western blots show ENGase and GAPDH expressions in synovial fibroblast extracts. Graphics represent average of protein expression from three different experiments done with synovial fibroblasts from three different OA patients. Values were calculated as in Figure 1 . a * and b * are significantly higher than controls. a * is significantly lower from b * . ( B ) p62-HA or p62-HA mutant S349A that has no phosphorylation on Serine 349 or p62-HA mutant S349E that has constitutive mimetic phosphorylation on Serine 349 (8) are co-transfected with NGLY1 or pEGFP-C1 into HEK293 cells. Western blots show P-S349 p62, p62, GFP, NGLY1 and GAPDH expressions in HEK293 extracts.
Figure Legend Snippet: Starvation increases ENGase turnover in synovial fibroblasts Co-transfection of NGLY1 with p62 increases p62 expression in HEK293 cells. ( A ) Synovial fibroblasts were cultured with or without serum, in the presence of different concentrations of MG132 as indicated. Western blots show ENGase and GAPDH expressions in synovial fibroblast extracts. Graphics represent average of protein expression from three different experiments done with synovial fibroblasts from three different OA patients. Values were calculated as in Figure 1 . a * and b * are significantly higher than controls. a * is significantly lower from b * . ( B ) p62-HA or p62-HA mutant S349A that has no phosphorylation on Serine 349 or p62-HA mutant S349E that has constitutive mimetic phosphorylation on Serine 349 (8) are co-transfected with NGLY1 or pEGFP-C1 into HEK293 cells. Western blots show P-S349 p62, p62, GFP, NGLY1 and GAPDH expressions in HEK293 extracts.

Techniques Used: Cotransfection, Expressing, Cell Culture, Western Blot, Mutagenesis, Transfection

40) Product Images from "Regions of the Herpes Simplex Virus Type 1 Latency-Associated Transcript That Protect Cells from Apoptosis In Vitro and Protect Neuronal Cells In Vivo"

Article Title: Regions of the Herpes Simplex Virus Type 1 Latency-Associated Transcript That Protect Cells from Apoptosis In Vitro and Protect Neuronal Cells In Vivo

Journal: Journal of Virology

doi: 10.1128/JVI.76.2.717-729.2002

In vitro inhibition of apoptosis by LAT in HeLa cells (A) and in neuron-like SY5Y cells (B). Cells were transfected with 1 μg of pEGFP-C1 (GFP-expressing construct) and 3 μg of pcDNA3 vector, pcDNA3. Pst-Mlu , or pCIp35 expressing the baculovirus antiapoptotic protein. At 48 h posttransfection, anti-Fas antibody was added to HeLa cells, while camptothecin was added to SY5Y cultures. At various times after treatment, GFP-positive cells were identified under a fluorescent microscope. ✻, positive control. The number of GFP-positive cells in control untreated cells represents 100% survival. Data are averages from five separate experiments.
Figure Legend Snippet: In vitro inhibition of apoptosis by LAT in HeLa cells (A) and in neuron-like SY5Y cells (B). Cells were transfected with 1 μg of pEGFP-C1 (GFP-expressing construct) and 3 μg of pcDNA3 vector, pcDNA3. Pst-Mlu , or pCIp35 expressing the baculovirus antiapoptotic protein. At 48 h posttransfection, anti-Fas antibody was added to HeLa cells, while camptothecin was added to SY5Y cultures. At various times after treatment, GFP-positive cells were identified under a fluorescent microscope. ✻, positive control. The number of GFP-positive cells in control untreated cells represents 100% survival. Data are averages from five separate experiments.

Techniques Used: In Vitro, Inhibition, Transfection, Expressing, Construct, Plasmid Preparation, Microscopy, Positive Control

Inhibition of caspase 8-induced apoptosis by LAT in vitro. HeLa cells were transfected with 3 μg of pcDNA3, pcDNA3. Pst-Mlu , or pICp35 together with 1 μg of pEGFP-C1 and 1 μg of the plasmid expressing caspase 8 (pC8). The pC8 plasmid was not transfected into control cells. (A) At 24 h after transfection, GFP-positive cells were visualized under a fluorescent scope and photographed. (B) GFP-positive cells were counted at 24, 48, and 72 h posttransfection, and data are expressed as the percentage of surviving GFP-positive cells over the control cells.
Figure Legend Snippet: Inhibition of caspase 8-induced apoptosis by LAT in vitro. HeLa cells were transfected with 3 μg of pcDNA3, pcDNA3. Pst-Mlu , or pICp35 together with 1 μg of pEGFP-C1 and 1 μg of the plasmid expressing caspase 8 (pC8). The pC8 plasmid was not transfected into control cells. (A) At 24 h after transfection, GFP-positive cells were visualized under a fluorescent scope and photographed. (B) GFP-positive cells were counted at 24, 48, and 72 h posttransfection, and data are expressed as the percentage of surviving GFP-positive cells over the control cells.

Techniques Used: Inhibition, In Vitro, Transfection, Plasmid Preparation, Expressing

Related Articles

Clone Assay:

Article Title: Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension
Article Snippet: .. Plasmids containing cloned sequences from the subtracted library were identified using a colony PCR protocol based on the PCR-Select Differential Screening protocol (Clontech). ..

Article Title: An Invasion-Independent Pathway of Blood-Borne Metastasis
Article Snippet: .. Plasmids containing cloned sequences from the subtracted libraries were identified using the PCR-Select Differential Screening protocol (Clontech). cDNA fragments consistently expressed at higher levels in MCH66 compared to MCH66C8 and MCH416 were sequenced using the Dye Terminator Cycle Sequencing Kit and automated fluorescent DNA sequencer (Amersham Pharmacia Biotech). ..

Article Title: Differentially Expressed Genes in Hormone Refractory Prostate Cancer
Article Snippet: .. Plasmids containing cloned sequences from each subtracted library were identified using a colony PCR protocol based on the PCR-Select Differential Screening protocol (Clontech). .. Briefly, inserts were amplified from each clone, 1× AmpliTaq Gold PCR buffer (10 mmol/L Tris (pH 9.0), 50 mmol/L KCl, 0.01% gelatin, 0.1% Triton X-100; Perkin Elmer, Foster City, CA), 1.5 mmol/L MgCl2 , 500 mmol/L, 200 μmol/L dNTPs, 0.3 μmol/L each nested primer (NP1 and NP2R) (Table 1) and 2.5U AmpliTaq Gold polymerase (Perkin Elmer) in a final volume of 20 μl.

Amplification:

Article Title: P311 induces a TGF-?1-independent, nonfibrogenic myofibroblast phenotype
Article Snippet: .. The mRNA from the two cultures was amplified using the SMART cDNA synthesis kit (CLONTECH Laboratories Inc., Palo Alto, California, USA), and PCR-Select (CLONTECH Laboratories Inc.) was then used for suppressive subtraction hybridization. ..

Random Primed:

Article Title: Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension
Article Snippet: .. The membranes were hybridised with random primed 32 P-dCTP labelled probes from either the subtracted or unsubtracted library as described in the PCR-select differential screening protocol (Clontech) and hybridised overnight at 72°C. ..

Polymerase Chain Reaction:

Article Title: Memory consolidation and gene expression in Periplaneta americana
Article Snippet: .. Hybridization and washing steps were performed as described in the PCR-Select Differential Screening protocol (Clontech). .. A peroxidase conjugated anti-DIG antibody (Boehringer) and Supersignal west femto chemiluminescent substrate (Pierce) combination was used to visualize the hybridization signal on the cDNA arrays.

Article Title: Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension
Article Snippet: .. Plasmids containing cloned sequences from the subtracted library were identified using a colony PCR protocol based on the PCR-Select Differential Screening protocol (Clontech). ..

Article Title: Differentially Expressed Genes in Hormone Refractory Prostate Cancer
Article Snippet: .. Probes for both subtracted and unsubtracted libraries from both LNCaP-r and LNCaP cell lines were prepared as described in the PCR-Select Differential Screening protocol (Clontech). .. RNA dot blot membranes were prehybridized in Express Hybridization solution (Clontech) supplemented with 0.2× SSC, 0.01 volume of blocking solution (100 μg/ml denatured sheared salmon sperm DNA and 3 μg/ml primers NP1 and NP2R) (Table 1) at 72°C for > 60 minutes.

Article Title: An Invasion-Independent Pathway of Blood-Borne Metastasis
Article Snippet: .. Plasmids containing cloned sequences from the subtracted libraries were identified using the PCR-Select Differential Screening protocol (Clontech). cDNA fragments consistently expressed at higher levels in MCH66 compared to MCH66C8 and MCH416 were sequenced using the Dye Terminator Cycle Sequencing Kit and automated fluorescent DNA sequencer (Amersham Pharmacia Biotech). ..

Article Title: P311 induces a TGF-?1-independent, nonfibrogenic myofibroblast phenotype
Article Snippet: .. The mRNA from the two cultures was amplified using the SMART cDNA synthesis kit (CLONTECH Laboratories Inc., Palo Alto, California, USA), and PCR-Select (CLONTECH Laboratories Inc.) was then used for suppressive subtraction hybridization. ..

Article Title: Differentially Expressed Genes in Hormone Refractory Prostate Cancer
Article Snippet: .. Plasmids containing cloned sequences from each subtracted library were identified using a colony PCR protocol based on the PCR-Select Differential Screening protocol (Clontech). .. Briefly, inserts were amplified from each clone, 1× AmpliTaq Gold PCR buffer (10 mmol/L Tris (pH 9.0), 50 mmol/L KCl, 0.01% gelatin, 0.1% Triton X-100; Perkin Elmer, Foster City, CA), 1.5 mmol/L MgCl2 , 500 mmol/L, 200 μmol/L dNTPs, 0.3 μmol/L each nested primer (NP1 and NP2R) (Table 1) and 2.5U AmpliTaq Gold polymerase (Perkin Elmer) in a final volume of 20 μl.

Article Title: Upregulated Genes In Sporadic, Idiopathic Pulmonary Arterial Hypertension
Article Snippet: .. The membranes were hybridised with random primed 32 P-dCTP labelled probes from either the subtracted or unsubtracted library as described in the PCR-select differential screening protocol (Clontech) and hybridised overnight at 72°C. ..

Article Title: PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family
Article Snippet: .. When E1A– p53 −/− MEFs were treated with 0.2 μg/ml doxorubicin for ∼17–18 hr, they failed to undergo apoptosis, as expected (Fig. C). cDNA was made from poly(A)+ RNA derived from each population, and the driver population was subtracted from the tester population using a variant of representational difference analysis (RDA), termed PCR-Select (Clontech; see Materials and Methods). .. Cloned cDNAs were sequenced to determine their identities and were validated by probing Northern blots to confirm apoptosis-specific expression.

Sequencing:

Article Title: An Invasion-Independent Pathway of Blood-Borne Metastasis
Article Snippet: .. Plasmids containing cloned sequences from the subtracted libraries were identified using the PCR-Select Differential Screening protocol (Clontech). cDNA fragments consistently expressed at higher levels in MCH66 compared to MCH66C8 and MCH416 were sequenced using the Dye Terminator Cycle Sequencing Kit and automated fluorescent DNA sequencer (Amersham Pharmacia Biotech). ..

Variant Assay:

Article Title: PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family
Article Snippet: .. When E1A– p53 −/− MEFs were treated with 0.2 μg/ml doxorubicin for ∼17–18 hr, they failed to undergo apoptosis, as expected (Fig. C). cDNA was made from poly(A)+ RNA derived from each population, and the driver population was subtracted from the tester population using a variant of representational difference analysis (RDA), termed PCR-Select (Clontech; see Materials and Methods). .. Cloned cDNAs were sequenced to determine their identities and were validated by probing Northern blots to confirm apoptosis-specific expression.

Derivative Assay:

Article Title: PERP, an apoptosis-associated target of p53, is a novel member of the PMP-22/gas3 family
Article Snippet: .. When E1A– p53 −/− MEFs were treated with 0.2 μg/ml doxorubicin for ∼17–18 hr, they failed to undergo apoptosis, as expected (Fig. C). cDNA was made from poly(A)+ RNA derived from each population, and the driver population was subtracted from the tester population using a variant of representational difference analysis (RDA), termed PCR-Select (Clontech; see Materials and Methods). .. Cloned cDNAs were sequenced to determine their identities and were validated by probing Northern blots to confirm apoptosis-specific expression.

Hybridization:

Article Title: Memory consolidation and gene expression in Periplaneta americana
Article Snippet: .. Hybridization and washing steps were performed as described in the PCR-Select Differential Screening protocol (Clontech). .. A peroxidase conjugated anti-DIG antibody (Boehringer) and Supersignal west femto chemiluminescent substrate (Pierce) combination was used to visualize the hybridization signal on the cDNA arrays.

Article Title: P311 induces a TGF-?1-independent, nonfibrogenic myofibroblast phenotype
Article Snippet: .. The mRNA from the two cultures was amplified using the SMART cDNA synthesis kit (CLONTECH Laboratories Inc., Palo Alto, California, USA), and PCR-Select (CLONTECH Laboratories Inc.) was then used for suppressive subtraction hybridization. ..

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  • 91
    TaKaRa pegfp c1
    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected <t>pEGFP-C1</t> (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.
    Pegfp C1, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa plasmid cmv pegfp c1
    Expression of EGFP protein in cells transiently transfected with EGFP under the control of PABP3 promoter sequences. PABP3 genomic sequences extending from nt –498 to +30 were amplified by PCR, as described in Materials and Methods, and then inserted into a promoter-less EGFP vector <t>(pEGFP-1).</t> These constructs were transiently transfected into HeLa (black boxes) or NTERA-2 (grey boxes) cells. The numbers of cells counted were identical in the two transfection assays. EGFP fluorescence driven by each construct was normalised to that obtained with the <t>CMV</t> <t>promoter/pEGFP-C1</t> vector (fluorescence of 100%). Results are the means of triplicate determinations obtained in two independent experiments. CMV corresponds to the control construct of the CMV promoter upstream from EGFP. PC, P1, P2 and P3 refer to the different PABP3 promoter constructs upstream from the EGFP reporter gene as indicated on the left.
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    TaKaRa egfp fused kctd5 encoding plasmid
    Retention of <t>KCTD5</t> in the Ni 2+ -NTA column during H. pylori infection. (A) HEK293T and (B) AGS cells were transfected with plasmids encoding <t>EGFP,</t> EGFP-KCTD5 and His-Ubi, lysates from these cells were processed for pull-down with Ni 2+ -NTA column, and the presence of KCTD5 was determined by immunoblot with antibody to KCTD5 (upper panel) and ubiquitinated proteins were shown as retention control in the column (lower panel). AGS transfected cells were infected by 8 h with H. pylori (MOI = 300).
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    Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Journal: PLoS ONE

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    doi: 10.1371/journal.pone.0043283

    Figure Lengend Snippet: Effects of co-transfected plasmids on expression of luciferase reporters. (A) Different plasmids have different effects on luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 150 ng of a tested plasmid. Renilla luciferase (RL) and firefly luciferase (FL) activities in pBS co-transfection were set to one. Data represent results of four transfection experiments performed in triplicates. Error bars = SEM. (B) Dose-dependent suppression of luciferase activities by co-transfected pEGFP-C1 (upper panel) and pRFP-T (lower panel). HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng/well of pEGFP-C1 or pRFP-T. The amount of transfected DNA was kept constant by adding pBS. Error bars = SEM. Data represent results of four transfection experiments performed in triplicates. (C) pEGFP-C1 negatively affects RFP reporter expression. HEK-293 cells were co-transfected with 150 ng/well of pCI-RFPT plasmid and 350 ng/well of pBS or pEGFP-C1 plasmid. RFP expression was analyzed 36 hours post-transfection by flow cytometry. X axis = RFP fluorescence intensity. Y axis = cell count. Colored curves show distribution of RFP signal as follows: black curve = untransfected cells; blue curve = pCI-RFPT + pBS co-transfection, and red curve = pCI-RFPT + pEGFP-C1 co-transfection. Total counts of transfected (RFP-positive) cells were identical in both samples (Fig. S1C). The shape of the red curve suggests that pEGFP-C1 reduces RFP fluorescence in transfected cells. The experiment has been performed three times, results from a representative experiment are shown.

    Article Snippet: To get further insights into possible causes of inhibitory effects of pEGFP-C1, we re-examined deep sequencing data searching for any transcriptome features unique to pEGFP-C1.

    Techniques: Transfection, Expressing, Luciferase, Plasmid Preparation, Cotransfection, Flow Cytometry, Cytometry, Fluorescence, Cell Counting

    Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Journal: PLoS ONE

    Article Title: Deep Sequencing Reveals Complex Spurious Transcription from Transiently Transfected Plasmids

    doi: 10.1371/journal.pone.0043283

    Figure Lengend Snippet: Kan/Neo cassette has a unique small RNA signature and contributes to downregulated expression of luciferase reporters. (A) Analysis of putative adenosine-deaminated small RNAs derived from Kan/Neo cassette (left panel) and pBS (right panel). The distribution of 20–24 nt reads with A/G conversions along pEGFP-C1 and pBS sequences is shown. (B) Size distribution of RNAs originating from EGFP CDS and Kan/Neo CDS sequences in HEK-293 cells. Small RNAs are sorted along the X-axis according to their length (18–26 nt long reads are shown). The Y-axis in both graphs shows the absolute number of reads carrying EGFP- (left) or Kan/Neo-derived sequences (right). The gray portion of each column indicates the fraction of reads carrying up to five A/G sequence changes. Note the absence of edited reads from EGFP CDS region. (C) Replacement of the Kan/Neo cassette by Amp r (denoted by _Amp) relieves repression of luciferase reporters. HEK-293 cells were co-transfected with 100 ng/well of each luciferase reporter and 0–250 ng of one of the four plasmids shown above the graph. The total amount of transfected DNA was kept constant by adding pBS. Renilla luciferase activity relative to the sample co-transfected with pBS (dashed line) is shown. Error bars = SEM. Data represent two independent experiments done in quadruplicates.

    Article Snippet: To get further insights into possible causes of inhibitory effects of pEGFP-C1, we re-examined deep sequencing data searching for any transcriptome features unique to pEGFP-C1.

    Techniques: Expressing, Luciferase, Derivative Assay, Sequencing, Transfection, Activity Assay

    Expression of EGFP protein in cells transiently transfected with EGFP under the control of PABP3 promoter sequences. PABP3 genomic sequences extending from nt –498 to +30 were amplified by PCR, as described in Materials and Methods, and then inserted into a promoter-less EGFP vector (pEGFP-1). These constructs were transiently transfected into HeLa (black boxes) or NTERA-2 (grey boxes) cells. The numbers of cells counted were identical in the two transfection assays. EGFP fluorescence driven by each construct was normalised to that obtained with the CMV promoter/pEGFP-C1 vector (fluorescence of 100%). Results are the means of triplicate determinations obtained in two independent experiments. CMV corresponds to the control construct of the CMV promoter upstream from EGFP. PC, P1, P2 and P3 refer to the different PABP3 promoter constructs upstream from the EGFP reporter gene as indicated on the left.

    Journal: Nucleic Acids Research

    Article Title: Human testis expresses a specific poly(A)-binding protein

    doi:

    Figure Lengend Snippet: Expression of EGFP protein in cells transiently transfected with EGFP under the control of PABP3 promoter sequences. PABP3 genomic sequences extending from nt –498 to +30 were amplified by PCR, as described in Materials and Methods, and then inserted into a promoter-less EGFP vector (pEGFP-1). These constructs were transiently transfected into HeLa (black boxes) or NTERA-2 (grey boxes) cells. The numbers of cells counted were identical in the two transfection assays. EGFP fluorescence driven by each construct was normalised to that obtained with the CMV promoter/pEGFP-C1 vector (fluorescence of 100%). Results are the means of triplicate determinations obtained in two independent experiments. CMV corresponds to the control construct of the CMV promoter upstream from EGFP. PC, P1, P2 and P3 refer to the different PABP3 promoter constructs upstream from the EGFP reporter gene as indicated on the left.

    Article Snippet: Plasmid CMV/pEGFP-C1 (Clontech), which contains the human cytomegalovirus (CMV) promoter, was used as a positive control in transfection experiments.

    Techniques: Expressing, Transfection, Genomic Sequencing, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Construct, Fluorescence

    Retention of KCTD5 in the Ni 2+ -NTA column during H. pylori infection. (A) HEK293T and (B) AGS cells were transfected with plasmids encoding EGFP, EGFP-KCTD5 and His-Ubi, lysates from these cells were processed for pull-down with Ni 2+ -NTA column, and the presence of KCTD5 was determined by immunoblot with antibody to KCTD5 (upper panel) and ubiquitinated proteins were shown as retention control in the column (lower panel). AGS transfected cells were infected by 8 h with H. pylori (MOI = 300).

    Journal: Frontiers in Cellular and Infection Microbiology

    Article Title: KCTD5 and Ubiquitin Proteasome Signaling Are Required for Helicobacter pylori Adherence

    doi: 10.3389/fcimb.2017.00450

    Figure Lengend Snippet: Retention of KCTD5 in the Ni 2+ -NTA column during H. pylori infection. (A) HEK293T and (B) AGS cells were transfected with plasmids encoding EGFP, EGFP-KCTD5 and His-Ubi, lysates from these cells were processed for pull-down with Ni 2+ -NTA column, and the presence of KCTD5 was determined by immunoblot with antibody to KCTD5 (upper panel) and ubiquitinated proteins were shown as retention control in the column (lower panel). AGS transfected cells were infected by 8 h with H. pylori (MOI = 300).

    Article Snippet: EGFP-fused KCTD5 encoding plasmid (pEGFP-C1-KCTD5) was generated by cloning the human KCTD5 cDNA from the pMaxKoz-HA-KCTD5 plasmid (Dementieva et al., ) (kindly donated by Dr. Steve Goldstein) into the XhoI/BamHI sites of the pEGFP-C1 plasmid (Catalog number: 6084, Clontech Laboratories, Mountain View, CA, USA).

    Techniques: Infection, Transfection