Structured Review

Thermo Fisher pcdna3 1
hPTTG1/GEF-H1 signalling regulates actin cytoskeleton rearrangements in breast cancer cells. ( A ) MDA-MB-231 cells with GFP or hPTTG1 knockdown or with hPTTG1 knockdown plus GEF-H1 overexpression were immunostained with anti-paxillin antibody (green) and phalloidin antibody (red) to observe focal adhesions and F-actin stress fibres, respectively. Depletion of hPTTG1 expression in MDA-MB-231 reduced the formation of focal adhesions (panel d), and F-actin stress fibres (panel e) when compared with control shGFP-knockdown MDA-MB-231 cells (panels a, b). Ectopic expression of GEF-H1 in hPTTG1-knockdown MDA-MB-231 cells increased punctate paxillin staining (panel g) and F-actin stress fibres (panel h). ( B ) Ectopic expression of hPTTG1 and GEF-H1 in MCF-7 cells induces the formation of focal adhesions (panels d, g), and F-actin stress fibres (panels e, h) when compared with control MCF-7 cells transfected with <t>pcDNA3.1</t> vector (Mock; panels a, b). ( C ) hPTTG1-overexpressed MCF-7 cells were plated on poly- L -lysine-coated coverslips for 48 h and then treated with C3 transferase (RhoA inhibitor, 2 μg/ml) or 555550 (ROCK inhibitor, 10 μ M ) for 4 or 1.5 h, respectively. The inhibitor-treated cells were then fixed and stained for DNA, focal adhesions (panels d, g) and F-actin (panels e, h). Both inhibitors impaired the phenotypes induced by hPTTG1. The vehicle-treated cells were shown in panels a–c. Scale bars, 20 μm. Merged images of paxillin and F-actin were shown in panels c, f and i of A -- C . DAPI (blue) was used to visualize the nuclei.
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1) Product Images from "Overexpressed hPTTG1 promotes breast cancer cell invasion and metastasis by regulating GEF-H1/RhoA signalling"

Article Title: Overexpressed hPTTG1 promotes breast cancer cell invasion and metastasis by regulating GEF-H1/RhoA signalling

Journal: Oncogene

doi: 10.1038/onc.2011.476

hPTTG1/GEF-H1 signalling regulates actin cytoskeleton rearrangements in breast cancer cells. ( A ) MDA-MB-231 cells with GFP or hPTTG1 knockdown or with hPTTG1 knockdown plus GEF-H1 overexpression were immunostained with anti-paxillin antibody (green) and phalloidin antibody (red) to observe focal adhesions and F-actin stress fibres, respectively. Depletion of hPTTG1 expression in MDA-MB-231 reduced the formation of focal adhesions (panel d), and F-actin stress fibres (panel e) when compared with control shGFP-knockdown MDA-MB-231 cells (panels a, b). Ectopic expression of GEF-H1 in hPTTG1-knockdown MDA-MB-231 cells increased punctate paxillin staining (panel g) and F-actin stress fibres (panel h). ( B ) Ectopic expression of hPTTG1 and GEF-H1 in MCF-7 cells induces the formation of focal adhesions (panels d, g), and F-actin stress fibres (panels e, h) when compared with control MCF-7 cells transfected with pcDNA3.1 vector (Mock; panels a, b). ( C ) hPTTG1-overexpressed MCF-7 cells were plated on poly- L -lysine-coated coverslips for 48 h and then treated with C3 transferase (RhoA inhibitor, 2 μg/ml) or 555550 (ROCK inhibitor, 10 μ M ) for 4 or 1.5 h, respectively. The inhibitor-treated cells were then fixed and stained for DNA, focal adhesions (panels d, g) and F-actin (panels e, h). Both inhibitors impaired the phenotypes induced by hPTTG1. The vehicle-treated cells were shown in panels a–c. Scale bars, 20 μm. Merged images of paxillin and F-actin were shown in panels c, f and i of A -- C . DAPI (blue) was used to visualize the nuclei.
Figure Legend Snippet: hPTTG1/GEF-H1 signalling regulates actin cytoskeleton rearrangements in breast cancer cells. ( A ) MDA-MB-231 cells with GFP or hPTTG1 knockdown or with hPTTG1 knockdown plus GEF-H1 overexpression were immunostained with anti-paxillin antibody (green) and phalloidin antibody (red) to observe focal adhesions and F-actin stress fibres, respectively. Depletion of hPTTG1 expression in MDA-MB-231 reduced the formation of focal adhesions (panel d), and F-actin stress fibres (panel e) when compared with control shGFP-knockdown MDA-MB-231 cells (panels a, b). Ectopic expression of GEF-H1 in hPTTG1-knockdown MDA-MB-231 cells increased punctate paxillin staining (panel g) and F-actin stress fibres (panel h). ( B ) Ectopic expression of hPTTG1 and GEF-H1 in MCF-7 cells induces the formation of focal adhesions (panels d, g), and F-actin stress fibres (panels e, h) when compared with control MCF-7 cells transfected with pcDNA3.1 vector (Mock; panels a, b). ( C ) hPTTG1-overexpressed MCF-7 cells were plated on poly- L -lysine-coated coverslips for 48 h and then treated with C3 transferase (RhoA inhibitor, 2 μg/ml) or 555550 (ROCK inhibitor, 10 μ M ) for 4 or 1.5 h, respectively. The inhibitor-treated cells were then fixed and stained for DNA, focal adhesions (panels d, g) and F-actin (panels e, h). Both inhibitors impaired the phenotypes induced by hPTTG1. The vehicle-treated cells were shown in panels a–c. Scale bars, 20 μm. Merged images of paxillin and F-actin were shown in panels c, f and i of A -- C . DAPI (blue) was used to visualize the nuclei.

Techniques Used: Multiple Displacement Amplification, Over Expression, Expressing, Staining, Transfection, Plasmid Preparation

hPTTG1 regulates the expression of GEF-H1 and the activation of RhoA. ( a ) Immunoblot analysis of hPTTG1 and GEF-H1 expression in control (not transfected with any DNA), GFP-knockdown or hPTTG1-knockdown MDA-MB-231 cells. ( b ) Immunoblot analysis of hPTTG1 and GEF-H1 expression in MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid. ( c ) Immunoblot analysis of active RhoA (RhoA-GTP) and total in control (not transfected with any DNA), GFP-knockdown or hPTTG1-knockdown MDA-MB-231 cells. ( d ) Immnuoblot analysis of active RhoA (RhoA-GTP) and total RhoA in MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid. The relative intensity of protein was quantified by Quantity One software and normalised to β- actin and total RhoA.
Figure Legend Snippet: hPTTG1 regulates the expression of GEF-H1 and the activation of RhoA. ( a ) Immunoblot analysis of hPTTG1 and GEF-H1 expression in control (not transfected with any DNA), GFP-knockdown or hPTTG1-knockdown MDA-MB-231 cells. ( b ) Immunoblot analysis of hPTTG1 and GEF-H1 expression in MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid. ( c ) Immunoblot analysis of active RhoA (RhoA-GTP) and total in control (not transfected with any DNA), GFP-knockdown or hPTTG1-knockdown MDA-MB-231 cells. ( d ) Immnuoblot analysis of active RhoA (RhoA-GTP) and total RhoA in MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid. The relative intensity of protein was quantified by Quantity One software and normalised to β- actin and total RhoA.

Techniques Used: Expressing, Activation Assay, Transfection, Multiple Displacement Amplification, Plasmid Preparation, Software

hPTTG1 regulates cell migration and invasion through GEF-H1. ( a ) Migration and invasion assays in MDA-MB-231 cells with GFP or hPTTG1 knockdown (hPTTG1 knockdown efficiency is shown in Figure 2a ). ( b ) Migration and invasion assays in MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid (hPTTG1 overexpression efficiency is shown in Figure 2b ). ( c ) Migration and invasion assays and immunoblot analysis in MDA-MB-231 cells with GFP or GEF-H1 knockdown. ( d ) Migration and invasion assays and immunoblot analysis in MDA-MB-231 cells with GFP or hPTTG1 knockdown or with hPTTG1 knockdown plus GEF-H1 overexpression. The relative intensity of protein was quantified by Quantity One software and normalised to β- actin. Results are presented as mean±s.e.m. ( n =3). * P
Figure Legend Snippet: hPTTG1 regulates cell migration and invasion through GEF-H1. ( a ) Migration and invasion assays in MDA-MB-231 cells with GFP or hPTTG1 knockdown (hPTTG1 knockdown efficiency is shown in Figure 2a ). ( b ) Migration and invasion assays in MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid (hPTTG1 overexpression efficiency is shown in Figure 2b ). ( c ) Migration and invasion assays and immunoblot analysis in MDA-MB-231 cells with GFP or GEF-H1 knockdown. ( d ) Migration and invasion assays and immunoblot analysis in MDA-MB-231 cells with GFP or hPTTG1 knockdown or with hPTTG1 knockdown plus GEF-H1 overexpression. The relative intensity of protein was quantified by Quantity One software and normalised to β- actin. Results are presented as mean±s.e.m. ( n =3). * P

Techniques Used: Migration, Multiple Displacement Amplification, Transfection, Plasmid Preparation, Expressing, Over Expression, Software

hPTTG1 is a transcriptional activator of GEF-H1. ( a ) Immunoblot analysis of hPTTG1 and GEF-H1 expression in all eight cell lines. β-Actin was used as a loading control (top). The transcript level of GEF-H1 was analysed by real-time PCR. GAPDH was used as the loading control (bottom). ( b ) The GEF-H1 regulatory sequence contains three putative hPTTG1-binding elements (binding element 1: nucleotides −1158 to −1153; binding element 2: nucleotides −998 to −993; binding element 3: nucleotides −269 to −264) (top). hPTTG1 activates the GEF-H1 promoter in MCF-7 cells. The GEF-H1 promoter was co-transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid, and harvested for luciferase reporter assays (bottom). ( c ) A series of GEF-H1 promoter deletion mutants (fragments B and C, top) were used for luciferase reporter assays in MCF-7 cells (bottom). ( d ) A site-specific mutation (TTAACT AAAACT) was introduced in the hPTTG1-binding element 1 on the GEF-H1 promoter and was co-transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid in MCF-7 cells, and luciferase reporter assays were performed. ( e ) ChIP assays were performed in MDA-MB-231 cells with GFP or hPTTG1 knockdown (hPTTG1 knockdown efficiency is shown in Figure 2a ). The ChIP-qPCR data are expressed as the fold increase over the control (IgG) on the promoter region containing hPTTG1-binding elements 1, 2 and 3. ( f ) GEF-H1 mRNA expression was analysed by real-time PCR in MDA-MB-231 cells with GFP or PTTG1 knockdown, and in ( g ) MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1. Human GAPDH was used as the loading control. Results are presented as mean±s.e.m. ( n =3). *** P
Figure Legend Snippet: hPTTG1 is a transcriptional activator of GEF-H1. ( a ) Immunoblot analysis of hPTTG1 and GEF-H1 expression in all eight cell lines. β-Actin was used as a loading control (top). The transcript level of GEF-H1 was analysed by real-time PCR. GAPDH was used as the loading control (bottom). ( b ) The GEF-H1 regulatory sequence contains three putative hPTTG1-binding elements (binding element 1: nucleotides −1158 to −1153; binding element 2: nucleotides −998 to −993; binding element 3: nucleotides −269 to −264) (top). hPTTG1 activates the GEF-H1 promoter in MCF-7 cells. The GEF-H1 promoter was co-transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid, and harvested for luciferase reporter assays (bottom). ( c ) A series of GEF-H1 promoter deletion mutants (fragments B and C, top) were used for luciferase reporter assays in MCF-7 cells (bottom). ( d ) A site-specific mutation (TTAACT AAAACT) was introduced in the hPTTG1-binding element 1 on the GEF-H1 promoter and was co-transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1 expression plasmid in MCF-7 cells, and luciferase reporter assays were performed. ( e ) ChIP assays were performed in MDA-MB-231 cells with GFP or hPTTG1 knockdown (hPTTG1 knockdown efficiency is shown in Figure 2a ). The ChIP-qPCR data are expressed as the fold increase over the control (IgG) on the promoter region containing hPTTG1-binding elements 1, 2 and 3. ( f ) GEF-H1 mRNA expression was analysed by real-time PCR in MDA-MB-231 cells with GFP or PTTG1 knockdown, and in ( g ) MCF-7 cells transfected with pcDNA3.1 vector (Mock) or pcDNA3.1-hPTTG1. Human GAPDH was used as the loading control. Results are presented as mean±s.e.m. ( n =3). *** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Sequencing, Binding Assay, Transfection, Plasmid Preparation, Luciferase, Mutagenesis, Chromatin Immunoprecipitation, Multiple Displacement Amplification

2) Product Images from "Rabaptin-5?/rabaptin-4 serves as a linker between rab4 and ?1-adaptin in membrane recycling from endosomes"

Article Title: Rabaptin-5?/rabaptin-4 serves as a linker between rab4 and ?1-adaptin in membrane recycling from endosomes

Journal: The EMBO Journal

doi: 10.1093/emboj/cdg257

Fig. 4. γ 1 –σ 1 AP-1 subcomplex is retained on rabaptin-5α beads. ( A ) GST or GST–rabaptin-5α(301–592) were incubated with glutathione–Sepharose and a HeLa cell extract. The western blot of bound (B) and non-bound (NB) fractions was probed with antibodies against γ 1 , β 1/2 , σ 1 , µ 1 and clathrin. ( B ) IF of clathrin and rabaptin-5α in cells transfected with rabaptin-5α or co-transfected with rabaptin-5α and γ 1 -adaptin. Cells were permeabilized, fixed and labelled with a rabbit antibody against rabaptin-5α (green) and X-22 against clathrin (red). ( C ) HeLa cells were labelled with [ 35 S]methionine and the lysate was incubated with GST–rabaptin-5α(301–592). Bound material was eluted with glutathione. Eluate and cell lysate were incubated with antibodies against γ 1 (100/3), β 1/2 (100/1), µ 1 and σ 1 , and protein A beads. Immunoprecipitates were resolved by SDS–PAGE and analysed by phosphorimaging. To detect σ 1 in the GSH eluate, gels were exposed 10 times longer than for γ 1 -adaptin (σ1 contains four times less methionine residues than γ 1 -adaptin). ( D ) Rabaptin-5α and γ 1 -adaptin co-localize on endosomes in the absence of intact AP-1. µ 1 A–/– and µ 1 A+/+ fibroblasts were transfected with rabaptin-5α-pcDNA3.1His. For γ 1 -adaptin labelling in µ 1 A+/+ fibroblasts, cells were extracted with saponin before fixation. Note that γ 1 -adaptin in these cells is not only present on the structures containing rabaptin-5α (arrows), but also in the TGN area (arrow heads). Saponin treatment of µ 1 A–/– cells removed all γ 1 -adaptin labelling (not shown). Cells were fixed, and labelled with a rabbit antibody against rabaptin-5α (red) and mouse γ 1 -adaptin antibody (Transduction labs) (green). Bar, 10 µm.
Figure Legend Snippet: Fig. 4. γ 1 –σ 1 AP-1 subcomplex is retained on rabaptin-5α beads. ( A ) GST or GST–rabaptin-5α(301–592) were incubated with glutathione–Sepharose and a HeLa cell extract. The western blot of bound (B) and non-bound (NB) fractions was probed with antibodies against γ 1 , β 1/2 , σ 1 , µ 1 and clathrin. ( B ) IF of clathrin and rabaptin-5α in cells transfected with rabaptin-5α or co-transfected with rabaptin-5α and γ 1 -adaptin. Cells were permeabilized, fixed and labelled with a rabbit antibody against rabaptin-5α (green) and X-22 against clathrin (red). ( C ) HeLa cells were labelled with [ 35 S]methionine and the lysate was incubated with GST–rabaptin-5α(301–592). Bound material was eluted with glutathione. Eluate and cell lysate were incubated with antibodies against γ 1 (100/3), β 1/2 (100/1), µ 1 and σ 1 , and protein A beads. Immunoprecipitates were resolved by SDS–PAGE and analysed by phosphorimaging. To detect σ 1 in the GSH eluate, gels were exposed 10 times longer than for γ 1 -adaptin (σ1 contains four times less methionine residues than γ 1 -adaptin). ( D ) Rabaptin-5α and γ 1 -adaptin co-localize on endosomes in the absence of intact AP-1. µ 1 A–/– and µ 1 A+/+ fibroblasts were transfected with rabaptin-5α-pcDNA3.1His. For γ 1 -adaptin labelling in µ 1 A+/+ fibroblasts, cells were extracted with saponin before fixation. Note that γ 1 -adaptin in these cells is not only present on the structures containing rabaptin-5α (arrows), but also in the TGN area (arrow heads). Saponin treatment of µ 1 A–/– cells removed all γ 1 -adaptin labelling (not shown). Cells were fixed, and labelled with a rabbit antibody against rabaptin-5α (red) and mouse γ 1 -adaptin antibody (Transduction labs) (green). Bar, 10 µm.

Techniques Used: Incubation, Western Blot, Transfection, SDS Page, Transduction

Fig. 2. Rabaptin-5α–γ-adaptin complex is localized on endosomes. HeLa cells were transfected with rabaptin-5α-pcDNA3.1His (left panels) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (right panels). Transfected cells were labelled for rabaptin-5α (green) and γ 1 -adaptin (red). Note the co-localization of rabaptin-5α and γ 1 -adaptin in transfected cells, and the distinct localization of γ 1 -adaptin in non-transfected cells (arrow) ( A and A ′). Cells were incubated with Alexa594-Tf for 60 min at 37°C and subsequently labelled with anti-Xpress antibody and Alexa488-conjugated IgG ( B and B ′). The TGN marker TGN46 does not relocate to enlarged endosomes. Transfected cells were labelled for rabaptin-5α (red) and TGN46 (green) ( C and C ′). Bar, 10 µm.
Figure Legend Snippet: Fig. 2. Rabaptin-5α–γ-adaptin complex is localized on endosomes. HeLa cells were transfected with rabaptin-5α-pcDNA3.1His (left panels) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (right panels). Transfected cells were labelled for rabaptin-5α (green) and γ 1 -adaptin (red). Note the co-localization of rabaptin-5α and γ 1 -adaptin in transfected cells, and the distinct localization of γ 1 -adaptin in non-transfected cells (arrow) ( A and A ′). Cells were incubated with Alexa594-Tf for 60 min at 37°C and subsequently labelled with anti-Xpress antibody and Alexa488-conjugated IgG ( B and B ′). The TGN marker TGN46 does not relocate to enlarged endosomes. Transfected cells were labelled for rabaptin-5α (red) and TGN46 (green) ( C and C ′). Bar, 10 µm.

Techniques Used: Transfection, Incubation, Marker

Fig. 6. Transfected rabaptin-5α delays Tf recycling. ( A ) HeLa cells were transfected with rabaptin-5α-pcDNA3.1His or with rabaptin-5α-pcDNA3.1His and γ 1 -adaptin-pcDNA3. The cells were incubated with 15 µg/ml Alexa-Tf at 16°C for 30 min, and subsequently chased at 37°C. Cells were fixed after different periods of time and stained for rabaptin-5α. Quantitation of the fraction of rabaptin-5α-positive endosomes containing Alexa488-Tf at 0, 10 and 50 min of chase. Error bars denote the standard deviation ( n = 10). ( B ) Rab4 and γ 1 -adaptin interaction domains in rabaptin-5α are required to retard Tf recycling. HeLa cells were transfected with rabaptin-5α(1–390), rabaptin-5α(301–592), rabaptin-5α(1–592) and rabaptin-5α. The cells were subjected to the pulse–chase protocol as above, fixed and labelled with anti-Xpress followed by Alexa488-labelled anti-mouse IgG. Note that only the rabaptin-5α truncation containing both rab4- and γ 1 -adaptin-binding sites retarded Tf recycling. Bar, 10 µm.
Figure Legend Snippet: Fig. 6. Transfected rabaptin-5α delays Tf recycling. ( A ) HeLa cells were transfected with rabaptin-5α-pcDNA3.1His or with rabaptin-5α-pcDNA3.1His and γ 1 -adaptin-pcDNA3. The cells were incubated with 15 µg/ml Alexa-Tf at 16°C for 30 min, and subsequently chased at 37°C. Cells were fixed after different periods of time and stained for rabaptin-5α. Quantitation of the fraction of rabaptin-5α-positive endosomes containing Alexa488-Tf at 0, 10 and 50 min of chase. Error bars denote the standard deviation ( n = 10). ( B ) Rab4 and γ 1 -adaptin interaction domains in rabaptin-5α are required to retard Tf recycling. HeLa cells were transfected with rabaptin-5α(1–390), rabaptin-5α(301–592), rabaptin-5α(1–592) and rabaptin-5α. The cells were subjected to the pulse–chase protocol as above, fixed and labelled with anti-Xpress followed by Alexa488-labelled anti-mouse IgG. Note that only the rabaptin-5α truncation containing both rab4- and γ 1 -adaptin-binding sites retarded Tf recycling. Bar, 10 µm.

Techniques Used: Transfection, Incubation, Staining, Quantitation Assay, Standard Deviation, Pulse Chase, Binding Assay

Fig. 3. Rabaptin-5α and γ-adaptin co-localize to tubulo-vesicular membrane clusters. Ultrathin cryosections of HeLa cells transfected with rabaptin- 5α-pcDNA3.1His (A,C and E) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (B and D). Double labelling of rabaptin-5α (10 nm gold) and γ 1 -adaptin (15 nm gold). Rabaptin-5α is associated with clusters of vesicular tubular membranes, where it co-localized with endogenous ( A ) and overexpressed γ 1 -adaptin ( B ). The overall density and diameter of rabaptin-5α-positive membranes is reminiscent of recycling tubules. The dense cytosol between the membranes indicates the presence of high concentrations of cytosolic protein. Note that some of the membranes display a coating typical of the presence of clathrin (arrows in A and B). Double labelling of TGN46 (15 nm gold) and rabaptin-5α (10 nm gold) revealed that rabaptin-5α-positive membranes do not overlap with TGN membranes ( C ). Rabaptin-5α- (15 nm gold) positive membranes do not contain internalized BSA–5 nm gold. Arrows point to compartments that contain the endocytic marker 10 min after internalization ( D ). Double labelling of rabaptin-5α (15 nm gold) and clathrin (10 nm gold). Some of the membranes within or associated with rabaptin-5α-positive membranes also stained for clathrin (arrows). Note that the nearby endosomal vacuole (E) has a normal morphology ( E ). G = Golgi complex, L = lysosome. Bar, 200 nm.
Figure Legend Snippet: Fig. 3. Rabaptin-5α and γ-adaptin co-localize to tubulo-vesicular membrane clusters. Ultrathin cryosections of HeLa cells transfected with rabaptin- 5α-pcDNA3.1His (A,C and E) or with rabaptin-5α and γ 1 -adaptin-pcDNA3 (B and D). Double labelling of rabaptin-5α (10 nm gold) and γ 1 -adaptin (15 nm gold). Rabaptin-5α is associated with clusters of vesicular tubular membranes, where it co-localized with endogenous ( A ) and overexpressed γ 1 -adaptin ( B ). The overall density and diameter of rabaptin-5α-positive membranes is reminiscent of recycling tubules. The dense cytosol between the membranes indicates the presence of high concentrations of cytosolic protein. Note that some of the membranes display a coating typical of the presence of clathrin (arrows in A and B). Double labelling of TGN46 (15 nm gold) and rabaptin-5α (10 nm gold) revealed that rabaptin-5α-positive membranes do not overlap with TGN membranes ( C ). Rabaptin-5α- (15 nm gold) positive membranes do not contain internalized BSA–5 nm gold. Arrows point to compartments that contain the endocytic marker 10 min after internalization ( D ). Double labelling of rabaptin-5α (15 nm gold) and clathrin (10 nm gold). Some of the membranes within or associated with rabaptin-5α-positive membranes also stained for clathrin (arrows). Note that the nearby endosomal vacuole (E) has a normal morphology ( E ). G = Golgi complex, L = lysosome. Bar, 200 nm.

Techniques Used: Transfection, Marker, Staining

3) Product Images from "CDC42-Interacting Protein 4 Gene Is Down Trans-Regulated by HBV DNA polymerase Trans Activated Protein 1"

Article Title: CDC42-Interacting Protein 4 Gene Is Down Trans-Regulated by HBV DNA polymerase Trans Activated Protein 1

Journal: Iranian Journal of Public Health

doi:

Western blot analysis of transient expression of HBVDNAPTP1 in THP-1 cells. lysates from THP-1 cells (lane 1). lysates from THP-1 cells transfected with pcDNA3.1(-)/myc-His A (lane 2). lysates from THP-1 cells transfected with pcDNA3.1(-)/myc-His A-HBVDNAPTP1 (lane 3)
Figure Legend Snippet: Western blot analysis of transient expression of HBVDNAPTP1 in THP-1 cells. lysates from THP-1 cells (lane 1). lysates from THP-1 cells transfected with pcDNA3.1(-)/myc-His A (lane 2). lysates from THP-1 cells transfected with pcDNA3.1(-)/myc-His A-HBVDNAPTP1 (lane 3)

Techniques Used: Western Blot, Expressing, Transfection

Digestion Identification of pcDNA3.1(-)/myc-His A-HBVDNAPTP1. DNA Marker DL2,000 (lane M). pcDNA3.1(-)/myc-His A-HBVDNAPTP1 Digested by BamH I and Hind III (lane 1). pcDNA3.1(-)/myc-His A-HBVDNAPTP1 Digested by Bgl II (lane 2)
Figure Legend Snippet: Digestion Identification of pcDNA3.1(-)/myc-His A-HBVDNAPTP1. DNA Marker DL2,000 (lane M). pcDNA3.1(-)/myc-His A-HBVDNAPTP1 Digested by BamH I and Hind III (lane 1). pcDNA3.1(-)/myc-His A-HBVDNAPTP1 Digested by Bgl II (lane 2)

Techniques Used: Marker

Quantitative Real time RT-PCR analysis of CIP4 gene in THP-1 cells. The amplification plot of CIP4 gene ( Fig 4A ). The dissociation curve of CIP4 gene ( Fig 4B ). The mRNA level of CIP4 in the experiment group (transfected with pcDNA3.1(-)/myc-His A-HBVDNAPTP1) compared with the control group (transfected with pcDNA3.1(-)/myc-His A) was detected using quantitative Real time RT-PCR. Data is expressed as relative quantity ( Fig 4C ).
Figure Legend Snippet: Quantitative Real time RT-PCR analysis of CIP4 gene in THP-1 cells. The amplification plot of CIP4 gene ( Fig 4A ). The dissociation curve of CIP4 gene ( Fig 4B ). The mRNA level of CIP4 in the experiment group (transfected with pcDNA3.1(-)/myc-His A-HBVDNAPTP1) compared with the control group (transfected with pcDNA3.1(-)/myc-His A) was detected using quantitative Real time RT-PCR. Data is expressed as relative quantity ( Fig 4C ).

Techniques Used: Quantitative RT-PCR, Amplification, Transfection

Western blot analysis of CIP4 expression in THP-1 cells. THP-1 cells transfected with pcDNA3.1(-)/myc-His A (lane 1). THP-1 cells transfected with pcDNA3.1(-)/myc-His A-HBVDNAPTP1 (lane 2)
Figure Legend Snippet: Western blot analysis of CIP4 expression in THP-1 cells. THP-1 cells transfected with pcDNA3.1(-)/myc-His A (lane 1). THP-1 cells transfected with pcDNA3.1(-)/myc-His A-HBVDNAPTP1 (lane 2)

Techniques Used: Western Blot, Expressing, Transfection

4) Product Images from "Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar"

Article Title: Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar

Journal: Scientific Reports

doi: 10.1038/srep26023

The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Cell Culture

The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Plasmid Preparation, Staining, Expressing, Construct

5) Product Images from "Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar"

Article Title: Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar

Journal: Scientific Reports

doi: 10.1038/srep26023

The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Cell Culture

The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Plasmid Preparation, Staining, Expressing, Construct

6) Product Images from "Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar"

Article Title: Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar

Journal: Scientific Reports

doi: 10.1038/srep26023

The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Cell Culture

The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Plasmid Preparation, Staining, Expressing, Construct

7) Product Images from "Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar"

Article Title: Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar

Journal: Scientific Reports

doi: 10.1038/srep26023

The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Cell Culture

The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Plasmid Preparation, Staining, Expressing, Construct

8) Product Images from "Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar"

Article Title: Simultaneous deactivation of FAK and Src improves the pathology of hypertrophic scar

Journal: Scientific Reports

doi: 10.1038/srep26023

The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar fibroblasts with various FAK and/or Src constructs on the steady-state protein levels of Col I, Col III and α-SMA. ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src, p -Src-Tyr 529 , Col I, Col III and α-SMA following transfection of hypertrophic scar fibroblasts with eukaryotic expression vector pcDNA3.1(+) alone vs . various FAK or Src wide-type or mutational constructs. Fibroblasts cultured between 3 rd to 4 th sub-passages were transfected with plasmid DNA, cell lysates were harvested three days thereafter and subjected to immunoblotting. Actin served as an equal protein loading control. ( B–H ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ), p -Src-Tyr 529 ( E ), Col I ( F ), Col III ( G ) and□α-SMA□( H ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Construct, Expressing, Plasmid Preparation, Cell Culture

The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P
Figure Legend Snippet: The effect of transfection of hypertrophic scar tissue with various FAK and/or Src mutants on extracellular matrix deposition and collagen fiber arrangement. Hypertrophic scar tissues were transfected with plasmid DNA of pcDNA3.1(+) vector alone vs . various FAK and/or Src mutants. Seven days thereafter scar samples were either subjected to immunoblotting ( A ), or paraffin-embedded and sectioned at 4-μm and processed for H E ( F ) or Masson staining ( G ). ( A ) Immunoblotting analysis on the expression of FAK, p -FAK-Tyr 407 , Src and p -Src-Tyr 529 following transfection of various constructs. ( B–E ) Bar graphs summarize immunoblotting results of FAK ( B ), p -FAK-Tyr 407 ( C ), Src ( D ) and p -Src-Tyr 529 ( E ) from three independent experiments. Each data point was normalized against its corresponding actin level with the value in pcDNA3.1(+) alone group arbitrarily set as 1. Each bar represents a mean ± SD of n = 5. * P

Techniques Used: Transfection, Plasmid Preparation, Staining, Expressing, Construct

9) Product Images from "Viral Interferon Regulatory Factors Decrease the Induction of Type I and Type II Interferon during Rhesus Macaque Rhadinovirus Infection"

Article Title: Viral Interferon Regulatory Factors Decrease the Induction of Type I and Type II Interferon during Rhesus Macaque Rhadinovirus Infection

Journal: Journal of Virology

doi: 10.1128/JVI.05047-11

R6 vIRF inhibits IRF-3-mediated transcription. RRV vIRFs (R6, R7, R10, and R11) were C-terminally tagged with HA, cloned into pcDNA3.1(−) using oligonucleotides listed in , and transiently transfected. (A) Whole-cell lysates were collected
Figure Legend Snippet: R6 vIRF inhibits IRF-3-mediated transcription. RRV vIRFs (R6, R7, R10, and R11) were C-terminally tagged with HA, cloned into pcDNA3.1(−) using oligonucleotides listed in , and transiently transfected. (A) Whole-cell lysates were collected

Techniques Used: Clone Assay, Transfection

10) Product Images from "Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling *"

Article Title: Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.685578

ATF4 induced CSE transcription via direct binding to a cis regulatory intronic site. A and B , luciferase activity resulting from HEK cells transfected with constructs as indicated together with (empty vector control plasmid) pCDNA3.1 or overexpressed ATF4. RLU , relative light units. n = 4. *, p
Figure Legend Snippet: ATF4 induced CSE transcription via direct binding to a cis regulatory intronic site. A and B , luciferase activity resulting from HEK cells transfected with constructs as indicated together with (empty vector control plasmid) pCDNA3.1 or overexpressed ATF4. RLU , relative light units. n = 4. *, p

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

Nox4 regulated CSE expression via ATF4. A , representative Western blot and quantitative densitometric analyses of ATF4 protein expression and the corresponding QPCR analyses of CSE mRNA expression in HUVECs after 24-h ATF4 or control pCDNA3.1 overexpression. A.U. , absorbance units. B , representative Western blot and quantitative densitometric analyses of ATF4 protein expression in HUVEC after 48 h of treatment with ATF4-targeted siRNA (siATF4) or control siRNA (siScram) together with 24 h Nox4 or β-gal ( B Gal ) overexpression as indicated. C , QPCR analyses of ATF4 and CSE mRNA expression in HUVECs after treatments as in B. D , representative Western blot and corresponding densitometric analyses of ATF4 protein expression in CMECs isolated from WT and eNox4 Tg mice. All data are normalized to β-actin mRNA and protein expression. n = 3; *, p
Figure Legend Snippet: Nox4 regulated CSE expression via ATF4. A , representative Western blot and quantitative densitometric analyses of ATF4 protein expression and the corresponding QPCR analyses of CSE mRNA expression in HUVECs after 24-h ATF4 or control pCDNA3.1 overexpression. A.U. , absorbance units. B , representative Western blot and quantitative densitometric analyses of ATF4 protein expression in HUVEC after 48 h of treatment with ATF4-targeted siRNA (siATF4) or control siRNA (siScram) together with 24 h Nox4 or β-gal ( B Gal ) overexpression as indicated. C , QPCR analyses of ATF4 and CSE mRNA expression in HUVECs after treatments as in B. D , representative Western blot and corresponding densitometric analyses of ATF4 protein expression in CMECs isolated from WT and eNox4 Tg mice. All data are normalized to β-actin mRNA and protein expression. n = 3; *, p

Techniques Used: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Isolation, Mouse Assay

11) Product Images from "The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells"

Article Title: The deubiquitinating enzyme UCHL1 negatively regulates the immunosuppressive capacity and survival of multipotent mesenchymal stromal cells

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0532-y

UCHL1 overexpression inhibited the ability of murine MSCs to suppress T cell proliferation. a , b Murine MSCs were transfected with pcDNA3.1 (vector-MSCs) or pcDNA3.1 containing UCHL1 (UCHL1-MSCs), and UCHL1 expression was measured by immunoblotting analysis and quantitative real-time PCR. c Irradiated control MSCs, vector-MSCs and UCHL1-MSCs were co-cultured with CFSE-labeled splenocytes for 3 days in the presence of anti-CD3/CD28 antibodies at the indicated ratios. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P
Figure Legend Snippet: UCHL1 overexpression inhibited the ability of murine MSCs to suppress T cell proliferation. a , b Murine MSCs were transfected with pcDNA3.1 (vector-MSCs) or pcDNA3.1 containing UCHL1 (UCHL1-MSCs), and UCHL1 expression was measured by immunoblotting analysis and quantitative real-time PCR. c Irradiated control MSCs, vector-MSCs and UCHL1-MSCs were co-cultured with CFSE-labeled splenocytes for 3 days in the presence of anti-CD3/CD28 antibodies at the indicated ratios. CD8 + and CD4 + T cells were collected for proliferation analysis by flow cytometry at the end of co-culture and MFI of CFSE of T cells were shown. Values are shown as mean ± S.E.M. and statistical significance indicated as * P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Real-time Polymerase Chain Reaction, Irradiation, Cell Culture, Labeling, Flow Cytometry, Cytometry, Co-Culture Assay

12) Product Images from "IL-32α suppresses colorectal cancer development via TNFR1-mediated death signaling"

Article Title: IL-32α suppresses colorectal cancer development via TNFR1-mediated death signaling

Journal: Oncotarget

doi:

Effects of stable expression of IL-32α in SW620 cells on colon cancer cell growth and apoptotic signaling (A) SW620 cells were stably transfected with either the empty pcDNA3.1 vector (SW-pcDNA cells) or the IL-32α expression vector (SW-IL-32α cells), respectively. Cell growth rate was measured by MTT assay during 72 hr (B) Expression of IL-32α and TNFR1 is shown by Western blot analysis. β-actin protein was used as an loading control. (C) Cells were treated with 30 ng/ml TNFα for 24 hr. Cell extracts were analyzed by Western blotting using specific antibodies. (D) The cells were treated with TNFα (30 ng/ml) for the indicated times and assayed to detect phospho-JNK and JNK. The data are represented as relative percentages of the control. *Significant difference from SW-pcDNA cells (* p
Figure Legend Snippet: Effects of stable expression of IL-32α in SW620 cells on colon cancer cell growth and apoptotic signaling (A) SW620 cells were stably transfected with either the empty pcDNA3.1 vector (SW-pcDNA cells) or the IL-32α expression vector (SW-IL-32α cells), respectively. Cell growth rate was measured by MTT assay during 72 hr (B) Expression of IL-32α and TNFR1 is shown by Western blot analysis. β-actin protein was used as an loading control. (C) Cells were treated with 30 ng/ml TNFα for 24 hr. Cell extracts were analyzed by Western blotting using specific antibodies. (D) The cells were treated with TNFα (30 ng/ml) for the indicated times and assayed to detect phospho-JNK and JNK. The data are represented as relative percentages of the control. *Significant difference from SW-pcDNA cells (* p

Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, MTT Assay, Western Blot

13) Product Images from "3-Phosphoglycerate dehydrogenase expression is regulated by HOXA10 in murine endometrium and human endometrial cells"

Article Title: 3-Phosphoglycerate dehydrogenase expression is regulated by HOXA10 in murine endometrium and human endometrial cells

Journal: Reproduction (Cambridge, England)

doi: 10.1530/REP-08-0376

(A) HOXA10 protein expression in human cell lines and primary human endometrial cells. (a) Western blot results show that pcDNA/HOXA10 transfection induced HOXA10 protein expression in A2780 cells. (b) HOXA10 protein expression was increased after transfection with HOXA10 and decreased after transfection with HOXA10 siRNA in HESC. (c) Similarly, HOXA10 protein expression was increased after transfection with HOXA10 and decreased after transfection with HOXA10 siRNA in Ishikawa cells. (d) HOXA10 protein expression was increased by pcDNA3.1/HOXA10 transfection and was decreased by HOXA10 siRNA transfection in primary human endometrial cells. (e) HOXA10 protein expression was increased after transfection with pcDNA3.1/HOXA10 and decreased after transfection with HOXA10 siRNA in primary human epithelial cells. (B) HOXA10 regulated PHGDH protein expression in human cell lines and primary human endometrial cells. (a) Western blot results show that PHGDH protein expression was not changed in A2780 cells after pcDNA3.1/ HOXA10 and HOXA10 siRNA. (b) pcDNA/HOXA10 transfection decreased PHGDH protein expression and HOXA10 siRNA transfection increased PHGDH expression in HESC ells. (c) Western blot results also show that PHGDH protein expression was increased by HOXA10 siRNA transfection and decreased by HOXA10 expression in Ishikawa cells. (d) Similarly, western blot results show that PHGDH protein expression was increased by HOXA10 siRNA transfection and decreased by HOXA10 expression in primary human endometrial cells. (E) PHGDH protein expression was increased by HOXA10 siRNA transfection and decreased by HOXA10 expression in primary human epithelial cells.
Figure Legend Snippet: (A) HOXA10 protein expression in human cell lines and primary human endometrial cells. (a) Western blot results show that pcDNA/HOXA10 transfection induced HOXA10 protein expression in A2780 cells. (b) HOXA10 protein expression was increased after transfection with HOXA10 and decreased after transfection with HOXA10 siRNA in HESC. (c) Similarly, HOXA10 protein expression was increased after transfection with HOXA10 and decreased after transfection with HOXA10 siRNA in Ishikawa cells. (d) HOXA10 protein expression was increased by pcDNA3.1/HOXA10 transfection and was decreased by HOXA10 siRNA transfection in primary human endometrial cells. (e) HOXA10 protein expression was increased after transfection with pcDNA3.1/HOXA10 and decreased after transfection with HOXA10 siRNA in primary human epithelial cells. (B) HOXA10 regulated PHGDH protein expression in human cell lines and primary human endometrial cells. (a) Western blot results show that PHGDH protein expression was not changed in A2780 cells after pcDNA3.1/ HOXA10 and HOXA10 siRNA. (b) pcDNA/HOXA10 transfection decreased PHGDH protein expression and HOXA10 siRNA transfection increased PHGDH expression in HESC ells. (c) Western blot results also show that PHGDH protein expression was increased by HOXA10 siRNA transfection and decreased by HOXA10 expression in Ishikawa cells. (d) Similarly, western blot results show that PHGDH protein expression was increased by HOXA10 siRNA transfection and decreased by HOXA10 expression in primary human endometrial cells. (E) PHGDH protein expression was increased by HOXA10 siRNA transfection and decreased by HOXA10 expression in primary human epithelial cells.

Techniques Used: Expressing, Western Blot, Transfection

14) Product Images from "Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells"

Article Title: Cyclophilin B as a co-regulator of prolactin-induced gene expression and function in breast cancer cells

Journal: Journal of molecular endocrinology

doi: 10.1677/JME-09-0140

The effects of CypB overexpression and CypB knockdown on a Stat5-responsive reporter. (A) Luciferase assay using pGL4-CISH reporter. Cells were transfected with pGL4-CISH reporter, the renilla luciferase control (pGL4.73), and pcDNA3.1-CypB expression vector. Transfectants were cultured in the minimal defined medium for 24 h, followed by 24 h of PRL stimulation prior to luminescence assay. (B and C) CypB knockdown in T47D cells confirmed by real-time PCR and microarray (B), and transient transfection (C). (D and E) Luciferase assay using pGL4-CISH (D) and pGL4-LHRE (E). T47D parental cells (wt) or si-CypB cells were co-transfected with 100 ng pGL4-CISH (D) or pGL4-LHRE (E), along with 2 ng renilla luciferase control (pGL4.73) and 400 ng pcDNA3.1-CypB expression vector, and maintained in the FBS-containing growth medium overnight. Transfectants were then starved in the FBS-free minimal defined medium for 24 h, followed by 24 h of PRL (10 ng/ml for pGL4-CISH and 100 ng/ml for pGL4-LHRE) stimulation prior to luminescence assay. Statistical analysis was performed using two-way ANOVA. ** P
Figure Legend Snippet: The effects of CypB overexpression and CypB knockdown on a Stat5-responsive reporter. (A) Luciferase assay using pGL4-CISH reporter. Cells were transfected with pGL4-CISH reporter, the renilla luciferase control (pGL4.73), and pcDNA3.1-CypB expression vector. Transfectants were cultured in the minimal defined medium for 24 h, followed by 24 h of PRL stimulation prior to luminescence assay. (B and C) CypB knockdown in T47D cells confirmed by real-time PCR and microarray (B), and transient transfection (C). (D and E) Luciferase assay using pGL4-CISH (D) and pGL4-LHRE (E). T47D parental cells (wt) or si-CypB cells were co-transfected with 100 ng pGL4-CISH (D) or pGL4-LHRE (E), along with 2 ng renilla luciferase control (pGL4.73) and 400 ng pcDNA3.1-CypB expression vector, and maintained in the FBS-containing growth medium overnight. Transfectants were then starved in the FBS-free minimal defined medium for 24 h, followed by 24 h of PRL (10 ng/ml for pGL4-CISH and 100 ng/ml for pGL4-LHRE) stimulation prior to luminescence assay. Statistical analysis was performed using two-way ANOVA. ** P

Techniques Used: Over Expression, Luciferase, Chromogenic In Situ Hybridization, Transfection, Expressing, Plasmid Preparation, Cell Culture, Luminescence Assay, Real-time Polymerase Chain Reaction, Microarray

15) Product Images from "LncRNA Gas5 acts as a ceRNA to regulate PTEN expression by sponging miR-222-3p in papillary thyroid carcinoma"

Article Title: LncRNA Gas5 acts as a ceRNA to regulate PTEN expression by sponging miR-222-3p in papillary thyroid carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.23336

Gas5 controls the miR-222-3p’s target, PTEN ( A ) a: The binding site between miR-222-3p and the 3′-UTR of PTEN mRNA predicted by bioinformatics. b: Wild (PTEN-3′-UTR-WT) and mutant (PTEN-3′-UTR-MT) PTEN 3′-UTRs carried in recombinant luciferase mRNAs transcribed by RLuc-PTEN-3′-UTR-WT and RLuc-PTEN-3′- UTR-MT. The binding site was deleted from PTEN-3′-UTR-MT. ( B ) The luciferase reporter plasmid containing wild or mutant-type PTEN-3′-UTR was co-transfected into BHP5-16 and K1 cells with miR-222 -3p mimics in parallel with miR-nc. ( C ) The mRNA level of PTEN in BHP5-16 and K1 cells transfected with miR-222-3p-mimics or miR-nc. ( D ) PTEN protein levels were detected by western blot in BHP5-16 and K1 cells after transfection with miR-222-3p mimics or miR-222-3p inhibitor. Meanwhile, the total and phosphorylated AKT were examined by western blot. GAPDH was used as an internal control. Similar results were obtained in three independent experiments. ( E ) Western blot analysis of PTEN, total AKT and phosphorylated AKT protein level following treatment of BHP5-16 cells and K1 with pcDNA3.1/Gas5 vector. GAPDH was used as control. ( F ) Western blot analysis of PTEN, total AKT and phosphorylated AKT protein level following treatment of BHP5-16 cells with pcDNA3.1/Gas5 vector and miR-222-3p mimics. ( G ) Rluc-PTEN 3′-UTR-WT and miR-222-3p mimics were co-transfected into BHP5-16 and K1 cells with plasmids expressing pcDNA3.1/Gas5 vector or with a control vector to verify the ceRNA activity of Gas5. Histogram indicate values of luciferase measured 48h after transfection. Data represent the mean ± SD from three independent experiments. ** P
Figure Legend Snippet: Gas5 controls the miR-222-3p’s target, PTEN ( A ) a: The binding site between miR-222-3p and the 3′-UTR of PTEN mRNA predicted by bioinformatics. b: Wild (PTEN-3′-UTR-WT) and mutant (PTEN-3′-UTR-MT) PTEN 3′-UTRs carried in recombinant luciferase mRNAs transcribed by RLuc-PTEN-3′-UTR-WT and RLuc-PTEN-3′- UTR-MT. The binding site was deleted from PTEN-3′-UTR-MT. ( B ) The luciferase reporter plasmid containing wild or mutant-type PTEN-3′-UTR was co-transfected into BHP5-16 and K1 cells with miR-222 -3p mimics in parallel with miR-nc. ( C ) The mRNA level of PTEN in BHP5-16 and K1 cells transfected with miR-222-3p-mimics or miR-nc. ( D ) PTEN protein levels were detected by western blot in BHP5-16 and K1 cells after transfection with miR-222-3p mimics or miR-222-3p inhibitor. Meanwhile, the total and phosphorylated AKT were examined by western blot. GAPDH was used as an internal control. Similar results were obtained in three independent experiments. ( E ) Western blot analysis of PTEN, total AKT and phosphorylated AKT protein level following treatment of BHP5-16 cells and K1 with pcDNA3.1/Gas5 vector. GAPDH was used as control. ( F ) Western blot analysis of PTEN, total AKT and phosphorylated AKT protein level following treatment of BHP5-16 cells with pcDNA3.1/Gas5 vector and miR-222-3p mimics. ( G ) Rluc-PTEN 3′-UTR-WT and miR-222-3p mimics were co-transfected into BHP5-16 and K1 cells with plasmids expressing pcDNA3.1/Gas5 vector or with a control vector to verify the ceRNA activity of Gas5. Histogram indicate values of luciferase measured 48h after transfection. Data represent the mean ± SD from three independent experiments. ** P

Techniques Used: Binding Assay, Mutagenesis, Recombinant, Luciferase, Plasmid Preparation, Transfection, Western Blot, Expressing, Activity Assay

Gas5 represses proliferation of PTC cells in vitro and in vivo ( A ) QRT-PCR analysis of Gas5 expression level following treatment BHP5-16 and K1 cells with pcDNA3.1/Gas5 vector. ( B ) The proliferation of pcDNA3.1/Gas5 vector–transfected BHP5-16 and K1 cells were detected by MTT assay. ( C ) Colony formation assay was performed to determine the proliferation of pcDNA3.1/Gas5 vector–transfected BHP5-16 and K1 cells. Colonies were counted and captured. Data represent the mean ± SD from three independent experiments. ( D ) Tumor formation assay was conducted in female BALB/c nude mice by subcutaneous injection of BHP5-16 cells (5 × 10 5 ) transfected with pcDNA3.1/Gas5 or pcDNA3.1 into the back of the mice. After 6 weeks, tumor tissues were harvested and tumor volumes were measured. ** P
Figure Legend Snippet: Gas5 represses proliferation of PTC cells in vitro and in vivo ( A ) QRT-PCR analysis of Gas5 expression level following treatment BHP5-16 and K1 cells with pcDNA3.1/Gas5 vector. ( B ) The proliferation of pcDNA3.1/Gas5 vector–transfected BHP5-16 and K1 cells were detected by MTT assay. ( C ) Colony formation assay was performed to determine the proliferation of pcDNA3.1/Gas5 vector–transfected BHP5-16 and K1 cells. Colonies were counted and captured. Data represent the mean ± SD from three independent experiments. ( D ) Tumor formation assay was conducted in female BALB/c nude mice by subcutaneous injection of BHP5-16 cells (5 × 10 5 ) transfected with pcDNA3.1/Gas5 or pcDNA3.1 into the back of the mice. After 6 weeks, tumor tissues were harvested and tumor volumes were measured. ** P

Techniques Used: In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Plasmid Preparation, Transfection, MTT Assay, Colony Assay, Tube Formation Assay, Mouse Assay, Injection

MiR-222-3p promotes proliferation of PTC cells in vitro ( A ) Cell proliferation assay of BHP5-16 and K1 transfected with miR-222-3p mimics/inhibitor, the OD values were detected each day at the same time point by MTT assay. ( B ) Colony formation assays were performed to determine the proliferation of K1 cells transfected with miR-222-3p mimics, inhibitor or miR-222-3p mimics plus pcDNA3.1/Gas5. Data are expressed as mean ± SD from three independent experiments. * P
Figure Legend Snippet: MiR-222-3p promotes proliferation of PTC cells in vitro ( A ) Cell proliferation assay of BHP5-16 and K1 transfected with miR-222-3p mimics/inhibitor, the OD values were detected each day at the same time point by MTT assay. ( B ) Colony formation assays were performed to determine the proliferation of K1 cells transfected with miR-222-3p mimics, inhibitor or miR-222-3p mimics plus pcDNA3.1/Gas5. Data are expressed as mean ± SD from three independent experiments. * P

Techniques Used: In Vitro, Proliferation Assay, Transfection, MTT Assay

16) Product Images from "Design of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc02351c"

Article Title: Design of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc02351c

Journal: Chemical Science

doi: 10.1039/c5sc02351c

(a) Molecular structures of the fluorogenic probes FCANB and AcFCANB. (b) No-wash live cell imaging of protein labeling with AcFCANB and MBP (top) and MBP–PYP (bottom) expressed in HEK 293T cells. Scale bar: 10 μm. (c) Schematic illustration for the mechanism of labeling acceleration. (d) Structure of PYP showing the reactive Cys-69 surrounded by the four acidic residues targeted for mutation.
Figure Legend Snippet: (a) Molecular structures of the fluorogenic probes FCANB and AcFCANB. (b) No-wash live cell imaging of protein labeling with AcFCANB and MBP (top) and MBP–PYP (bottom) expressed in HEK 293T cells. Scale bar: 10 μm. (c) Schematic illustration for the mechanism of labeling acceleration. (d) Structure of PYP showing the reactive Cys-69 surrounded by the four acidic residues targeted for mutation.

Techniques Used: Live Cell Imaging, Labeling, Mutagenesis

17) Product Images from "Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription"

Article Title: Functionality of unliganded VDR in breast cancer cells: repressive action on CYP24 basal transcription

Journal: Molecular and cellular biochemistry

doi: 10.1007/s11010-010-0478-6

Effect of VDR Fok1 polymorphism on CYP24 promoter activity and CYP24 mRNA expression. a and b MCF-7 and MDA-MB231 cells were co-transfected with VDR expression vectors (containing the FokI site, VDRff, and VDRFF, respectively) or empty vector (pcDNA3.1)
Figure Legend Snippet: Effect of VDR Fok1 polymorphism on CYP24 promoter activity and CYP24 mRNA expression. a and b MCF-7 and MDA-MB231 cells were co-transfected with VDR expression vectors (containing the FokI site, VDRff, and VDRFF, respectively) or empty vector (pcDNA3.1)

Techniques Used: Activity Assay, Expressing, Multiple Displacement Amplification, Transfection, Plasmid Preparation

18) Product Images from "Monoclonal Antibodies Directed Against Chicken ?2-Microglobulin Developed With a Synthesized Peptide"

Article Title: Monoclonal Antibodies Directed Against Chicken ?2-Microglobulin Developed With a Synthesized Peptide

Journal: Monoclonal Antibodies in Immunodiagnosis and Immunotherapy

doi: 10.1089/mab.2013.0001

Indirect immunofluorescence imaging of MAb 6E7 binding to different cells. ( A ) HD11 cells (a); CEF cells (b); 293T/chβ2M (c); pcDNA3.1 transfected 293T cells (d). 200×( B ) chβ2M on the cell membrane and cytoplasm of HD11 cells (green,
Figure Legend Snippet: Indirect immunofluorescence imaging of MAb 6E7 binding to different cells. ( A ) HD11 cells (a); CEF cells (b); 293T/chβ2M (c); pcDNA3.1 transfected 293T cells (d). 200×( B ) chβ2M on the cell membrane and cytoplasm of HD11 cells (green,

Techniques Used: Immunofluorescence, Imaging, Binding Assay, Transfection

19) Product Images from "Increased cell apoptosis in human lung adenocarcinoma and in vivo tumor growth inhibition by RBM10, a tumor suppressor gene"

Article Title: Increased cell apoptosis in human lung adenocarcinoma and in vivo tumor growth inhibition by RBM10, a tumor suppressor gene

Journal: Oncology Letters

doi: 10.3892/ol.2017.6765

Evaluation of apoptosis-associated genes in cells transfected with pcDNA3.1 and pcDNA3.1-RBM10. (A) mRNA expression levels of RBM10, Bcl-2, cleaved caspase-9, cleaved caspase-3, PARP in CTRL cells and cells transfected with RBM10. The transfection of
Figure Legend Snippet: Evaluation of apoptosis-associated genes in cells transfected with pcDNA3.1 and pcDNA3.1-RBM10. (A) mRNA expression levels of RBM10, Bcl-2, cleaved caspase-9, cleaved caspase-3, PARP in CTRL cells and cells transfected with RBM10. The transfection of

Techniques Used: Transfection, Expressing

In vivo assessment of anti-tumor function of RBM10 in tumor-bearing BALB/c nude mice. The mice were transplanted with 5×10 5 A549 cells on day 0 to establish tumor formation. Salmonella carrying pcDNA3.1 or pcDNA3.1-RBM10 are injected every 2 days
Figure Legend Snippet: In vivo assessment of anti-tumor function of RBM10 in tumor-bearing BALB/c nude mice. The mice were transplanted with 5×10 5 A549 cells on day 0 to establish tumor formation. Salmonella carrying pcDNA3.1 or pcDNA3.1-RBM10 are injected every 2 days

Techniques Used: In Vivo, Mouse Assay, Injection

In vitro anti-tumor analysis of RBM10 mRNA levels in A549 cells. (A) The assessment of RBM10 mRNA and protein expression levels in A549 cells transfected with pcDNA3.1 (control) and pcDNA3.1-RBM10 and the relative expression levels of RBM10 mRNA and protein
Figure Legend Snippet: In vitro anti-tumor analysis of RBM10 mRNA levels in A549 cells. (A) The assessment of RBM10 mRNA and protein expression levels in A549 cells transfected with pcDNA3.1 (control) and pcDNA3.1-RBM10 and the relative expression levels of RBM10 mRNA and protein

Techniques Used: In Vitro, Expressing, Transfection

20) Product Images from "Lipocalin 2 decreases senescence of bone marrow-derived mesenchymal stem cells under sub-lethal doses of oxidative stress"

Article Title: Lipocalin 2 decreases senescence of bone marrow-derived mesenchymal stem cells under sub-lethal doses of oxidative stress

Journal: Cell Stress & Chaperones

doi: 10.1007/s12192-014-0496-5

Evaluation of Lcn2 mRNA expression in MSCs by real-time PCR. The ratio of Lcn2 to β-actin expression was evaluated in MSC-V and MSC-Lcn2. After transfection of the pcDNA3.1-Lcn2 plasmid, the expression of Lcn2 mRNA in MSCs was increased (data represents mean ± SD; number of replicates = 3, ** P
Figure Legend Snippet: Evaluation of Lcn2 mRNA expression in MSCs by real-time PCR. The ratio of Lcn2 to β-actin expression was evaluated in MSC-V and MSC-Lcn2. After transfection of the pcDNA3.1-Lcn2 plasmid, the expression of Lcn2 mRNA in MSCs was increased (data represents mean ± SD; number of replicates = 3, ** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation

21) Product Images from "SOX2 inhibits cell proliferation and metastasis, promotes apoptotic by downregulating CCND1 and PARP in gastric cancer"

Article Title: SOX2 inhibits cell proliferation and metastasis, promotes apoptotic by downregulating CCND1 and PARP in gastric cancer

Journal: American Journal of Translational Research

doi:

SOX2 overexpression inhibits the expression of Cyclin D1 and PARP in GC cells. A: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of Cyclin D1 mRNA was analyzed by qRT-PCR and 2 -ΔΔCT was shown. B: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of PARP mRNA was analyzed by qRT-PCR and 2 -ΔΔCT was shown. C: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The protein expression level of Cyclin D1 and PARP were analyzed by western blot. *P
Figure Legend Snippet: SOX2 overexpression inhibits the expression of Cyclin D1 and PARP in GC cells. A: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of Cyclin D1 mRNA was analyzed by qRT-PCR and 2 -ΔΔCT was shown. B: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of PARP mRNA was analyzed by qRT-PCR and 2 -ΔΔCT was shown. C: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The protein expression level of Cyclin D1 and PARP were analyzed by western blot. *P

Techniques Used: Over Expression, Expressing, Transfection, Quantitative RT-PCR, Western Blot

SOX2 overexpression inhibits GC cell migration and invasion. A, B: Representative migration or invasion images of HGC-27 and BGC-823 transfected with pcDNA3.1 or pcDNA3.1-SOX2. Cell numbers were counted in five random fields. *P
Figure Legend Snippet: SOX2 overexpression inhibits GC cell migration and invasion. A, B: Representative migration or invasion images of HGC-27 and BGC-823 transfected with pcDNA3.1 or pcDNA3.1-SOX2. Cell numbers were counted in five random fields. *P

Techniques Used: Over Expression, Migration, Transfection

SOX2 overexpression inhibits GC cell proliferation. A: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of SOX2 mRNA was analyzed by qRT-PCR and Log 10 (2 -ΔΔCT ) was shown. B: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of SOX2 protein was analyzed by western blot. C: The effects of SOX2 on the ability of proliferation of HGC-27 cells were analyzed by MTT assay. D: The effects of SOX2 on the ability of proliferation of BGC823 cells were analyzed by MTT assay. *P
Figure Legend Snippet: SOX2 overexpression inhibits GC cell proliferation. A: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of SOX2 mRNA was analyzed by qRT-PCR and Log 10 (2 -ΔΔCT ) was shown. B: HGC-27 and BGC-823 were transfected with pcDNA3.1 or pcDNA3.1-SOX2 for 48 h respectively. The expression of SOX2 protein was analyzed by western blot. C: The effects of SOX2 on the ability of proliferation of HGC-27 cells were analyzed by MTT assay. D: The effects of SOX2 on the ability of proliferation of BGC823 cells were analyzed by MTT assay. *P

Techniques Used: Over Expression, Transfection, Expressing, Quantitative RT-PCR, Western Blot, MTT Assay

22) Product Images from "CREB promotes laryngeal cancer cell migration via MYCT1/NAT10 axis"

Article Title: CREB promotes laryngeal cancer cell migration via MYCT1/NAT10 axis

Journal: OncoTargets and therapy

doi: 10.2147/OTT.S156582

CREB inhibits MYCT1 transcription. Notes: ( A ) Transcription factor CREB binding sites present in the MYCT1 promoter region. ( B ) MYCT1 promoter activity analysis. Hep2 cells were transfected with pGL3-P760 or pGL3-P795, followed by a luciferase reporter assay. Firefly luciferase activity was normalized to Renilla luciferase activity. ( C ) Effect of CREB on pGL3-P795 activity. pcDNA3.1 vector or pcDNA3.1-CREB was co-transfected with pGL3-P795 into Hep2 cells, followed by a luciferase reporter assay. ( D ) Effect of CREB on MYCT1 mRNA level. MYCT1 mRNA level was detected by qRT-PCR in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( E ) Effect of CREB on MYCT1 protein level. MYCT1 protein level was examined by Western blot in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( F ) Binding of CREB to MYCT1 in vivo. DNA fragments from Hep2 cells were amplified by PCR based on immunoprecipitation with anti-IgG or anti-CREB antibodies. ** P
Figure Legend Snippet: CREB inhibits MYCT1 transcription. Notes: ( A ) Transcription factor CREB binding sites present in the MYCT1 promoter region. ( B ) MYCT1 promoter activity analysis. Hep2 cells were transfected with pGL3-P760 or pGL3-P795, followed by a luciferase reporter assay. Firefly luciferase activity was normalized to Renilla luciferase activity. ( C ) Effect of CREB on pGL3-P795 activity. pcDNA3.1 vector or pcDNA3.1-CREB was co-transfected with pGL3-P795 into Hep2 cells, followed by a luciferase reporter assay. ( D ) Effect of CREB on MYCT1 mRNA level. MYCT1 mRNA level was detected by qRT-PCR in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( E ) Effect of CREB on MYCT1 protein level. MYCT1 protein level was examined by Western blot in Hep2 cells transfected with pcDNA3.1 or pcDNA3.1-CREB. ( F ) Binding of CREB to MYCT1 in vivo. DNA fragments from Hep2 cells were amplified by PCR based on immunoprecipitation with anti-IgG or anti-CREB antibodies. ** P

Techniques Used: Binding Assay, Activity Assay, Transfection, Luciferase, Reporter Assay, Plasmid Preparation, Quantitative RT-PCR, Western Blot, In Vivo, Amplification, Polymerase Chain Reaction, Immunoprecipitation

23) Product Images from "Long non-coding RNA MEG3 suppresses the development of bladder urothelial carcinoma by regulating miR-96 and TPM1"

Article Title: Long non-coding RNA MEG3 suppresses the development of bladder urothelial carcinoma by regulating miR-96 and TPM1

Journal: Cancer Biology & Therapy

doi: 10.1080/15384047.2018.1480279

MEG3 up-regulation suppressed cell proliferation and enhanced cell apoptosis. (A) RT-qPCR showed that pcDNA3.1-MEG3 had high transfection efficiency in 5637 cells and T-24 cells; (B) MTT assay demonstrated that pcDNA3.1-MEG3 could slow down cell proliferation in 5637 cells and T-24 cells; (C) Flow cytometry revealed that cell apoptosis of cells transfected with pcDNA3.1-MEG3 was high in 5637 cells and T-24 cells; (D) Flow cytometry showed that pcDNA3.1-MEG3 blocked cells in G0/G1 phase in 5637 cells and T-24 cells. * P
Figure Legend Snippet: MEG3 up-regulation suppressed cell proliferation and enhanced cell apoptosis. (A) RT-qPCR showed that pcDNA3.1-MEG3 had high transfection efficiency in 5637 cells and T-24 cells; (B) MTT assay demonstrated that pcDNA3.1-MEG3 could slow down cell proliferation in 5637 cells and T-24 cells; (C) Flow cytometry revealed that cell apoptosis of cells transfected with pcDNA3.1-MEG3 was high in 5637 cells and T-24 cells; (D) Flow cytometry showed that pcDNA3.1-MEG3 blocked cells in G0/G1 phase in 5637 cells and T-24 cells. * P

Techniques Used: Quantitative RT-PCR, Transfection, MTT Assay, Flow Cytometry, Cytometry

MEG3 and miR-96 suppression inhibited tumor growth by targeting TPM1 in vivo . (A) After injecting with pcDNA3.1-MEG3 or miR-96 inhibitor, tumor growths were decreased remarkably in vivo ; (B) Tumor volumes were examined. It was found out that tumor volume in pcDNA3.1-MEG3 group and miR-96 inhibitor group were smaller than in NC group; (C) Tumor weights were tested. It was found out that tumor weight in pcDNA3.1-MEG3 group and miR-96 inhibitor group were also lighter than in NC group; (D) Western blot showed that pcDNA3.1-MEG3 and miR-96 inhibitor could both up-regulated the expression of TPM1 . ** P
Figure Legend Snippet: MEG3 and miR-96 suppression inhibited tumor growth by targeting TPM1 in vivo . (A) After injecting with pcDNA3.1-MEG3 or miR-96 inhibitor, tumor growths were decreased remarkably in vivo ; (B) Tumor volumes were examined. It was found out that tumor volume in pcDNA3.1-MEG3 group and miR-96 inhibitor group were smaller than in NC group; (C) Tumor weights were tested. It was found out that tumor weight in pcDNA3.1-MEG3 group and miR-96 inhibitor group were also lighter than in NC group; (D) Western blot showed that pcDNA3.1-MEG3 and miR-96 inhibitor could both up-regulated the expression of TPM1 . ** P

Techniques Used: In Vivo, Western Blot, Expressing

TPM1 inhibited cell proliferation and promoted cell apoptosis in 5637 cells and T-24 cells. (A) RT-qPCR showed that miR-96 mimics, pcDNA3.1- TPM1 and pcDNA3.1- TPM1 + miR-96 mimics had high transfection efficiency in 5637 cells; (B) MTT assay demonstrated that cell proliferation in pcDNA3.1- TPM1 group was inhibited, while cell proliferation in miR-96 mimics group was accelerated. PcDNA3.1-MEG3+ miR-96 mimics group had no remarkable difference compared with NC group; (C) Flow cytometry revealed that pcDNA3.1- TPM1 could promote cell apoptosis, whereas miR-96 mimics inhibited it. PcDNA3.1-MEG3+ miR-96 mimics group had no remarkable difference compared with NC group; (D) Flow cytometry showed that cells transfected with pcDNA3.1- TPM1 were blocked in G0/G1 phase. But miR-96 mimics had the opposite effect. * P
Figure Legend Snippet: TPM1 inhibited cell proliferation and promoted cell apoptosis in 5637 cells and T-24 cells. (A) RT-qPCR showed that miR-96 mimics, pcDNA3.1- TPM1 and pcDNA3.1- TPM1 + miR-96 mimics had high transfection efficiency in 5637 cells; (B) MTT assay demonstrated that cell proliferation in pcDNA3.1- TPM1 group was inhibited, while cell proliferation in miR-96 mimics group was accelerated. PcDNA3.1-MEG3+ miR-96 mimics group had no remarkable difference compared with NC group; (C) Flow cytometry revealed that pcDNA3.1- TPM1 could promote cell apoptosis, whereas miR-96 mimics inhibited it. PcDNA3.1-MEG3+ miR-96 mimics group had no remarkable difference compared with NC group; (D) Flow cytometry showed that cells transfected with pcDNA3.1- TPM1 were blocked in G0/G1 phase. But miR-96 mimics had the opposite effect. * P

Techniques Used: Quantitative RT-PCR, Transfection, MTT Assay, Flow Cytometry, Cytometry

MEG3 inhibited cell proliferation and enhanced cell apoptosis by targeting miR-96 in 5637 cells and T-24 cells. (A) RT-qPCR showed that miR-96 mimics and miR-96 inhibitor had high transfection efficiency in 5637 cells; (B) MTT assay demonstrated that cell proliferation of cells transfected with pcDNA3.1-MEG3 or miR-96 inhibitor was slowed down, while cell proliferation of cells transfected with miR-96 mimics was accelerated. PcDNA3.1-MEG3+ miR-96 mimics group had no remarkable difference with NC group; (C) Flow cytometry revealed that pcDNA3.1-MEG3 and miR-96 inhibitor could promote cell apoptosis, whereas miR-96 mimics inhibited it. NC group also had no remarkable difference compared with pcDNA3.1-MEG3+ miR-96 mimics group. * P
Figure Legend Snippet: MEG3 inhibited cell proliferation and enhanced cell apoptosis by targeting miR-96 in 5637 cells and T-24 cells. (A) RT-qPCR showed that miR-96 mimics and miR-96 inhibitor had high transfection efficiency in 5637 cells; (B) MTT assay demonstrated that cell proliferation of cells transfected with pcDNA3.1-MEG3 or miR-96 inhibitor was slowed down, while cell proliferation of cells transfected with miR-96 mimics was accelerated. PcDNA3.1-MEG3+ miR-96 mimics group had no remarkable difference with NC group; (C) Flow cytometry revealed that pcDNA3.1-MEG3 and miR-96 inhibitor could promote cell apoptosis, whereas miR-96 mimics inhibited it. NC group also had no remarkable difference compared with pcDNA3.1-MEG3+ miR-96 mimics group. * P

Techniques Used: Quantitative RT-PCR, Transfection, MTT Assay, Flow Cytometry, Cytometry

TPM1 was a target gene of MiR-96 in 5637 cells and T-24 cells. (A) RT-qPCR showed that miR-96 and TPM1 expression in 45 paired tissues and revealed that miR-96 and TPM1 were negatively related with TPM1 ; (B) MiRBase predicted that TPM1 and miR-96 might have a binding site; (C) Dual luciferase reporter assay proved the direct target relationship. The group which binding site on TPM1 (mut group) was mutated had no significant difference after being treated with miR-96 mimics; (D) RT-qPCR showed that miR-96 mimics could down-regulate the expression of TPM1 ; (E) RT-qPCR demonstrated that pcDNA3.1- TPM1 could not down-regulate the production of miR-96; (F) Western blot revealed that PcDNA3.1- TPM1 group had high expressed TPM1 and miR-96 mimics group had low expressed TPM1 . PcDNA3.1- TPM1 + miR-96 mimics had no significant change compared with NC group. * P
Figure Legend Snippet: TPM1 was a target gene of MiR-96 in 5637 cells and T-24 cells. (A) RT-qPCR showed that miR-96 and TPM1 expression in 45 paired tissues and revealed that miR-96 and TPM1 were negatively related with TPM1 ; (B) MiRBase predicted that TPM1 and miR-96 might have a binding site; (C) Dual luciferase reporter assay proved the direct target relationship. The group which binding site on TPM1 (mut group) was mutated had no significant difference after being treated with miR-96 mimics; (D) RT-qPCR showed that miR-96 mimics could down-regulate the expression of TPM1 ; (E) RT-qPCR demonstrated that pcDNA3.1- TPM1 could not down-regulate the production of miR-96; (F) Western blot revealed that PcDNA3.1- TPM1 group had high expressed TPM1 and miR-96 mimics group had low expressed TPM1 . PcDNA3.1- TPM1 + miR-96 mimics had no significant change compared with NC group. * P

Techniques Used: Quantitative RT-PCR, Expressing, Binding Assay, Luciferase, Reporter Assay, Western Blot

MiR-96 was a target of MEG3 in 5637 cells and T-24 cells. (A) RT-qPCR showed that MEG3 and miR-96 expression in 45 paired tissues and revealed that MEG3 and miR-96 were negatively related; (B) MiRcode predicted that MEG3 and miR-96 might have a binding site; (C) Dual luciferase reporter assay proved that MEG3 bound to miR-96 directly. The group which binding site on MEG3 was mutated (mut group) had no significant difference after being treated with miR-96 mimics; (D) RT-qPCR demonstrated that pcDNA3.1-MEG3 could suppress the expression of miR-96; (E) RT-qPCR showed that miR-96 mimics could not affect the production of MEG3. * P
Figure Legend Snippet: MiR-96 was a target of MEG3 in 5637 cells and T-24 cells. (A) RT-qPCR showed that MEG3 and miR-96 expression in 45 paired tissues and revealed that MEG3 and miR-96 were negatively related; (B) MiRcode predicted that MEG3 and miR-96 might have a binding site; (C) Dual luciferase reporter assay proved that MEG3 bound to miR-96 directly. The group which binding site on MEG3 was mutated (mut group) had no significant difference after being treated with miR-96 mimics; (D) RT-qPCR demonstrated that pcDNA3.1-MEG3 could suppress the expression of miR-96; (E) RT-qPCR showed that miR-96 mimics could not affect the production of MEG3. * P

Techniques Used: Quantitative RT-PCR, Expressing, Binding Assay, Luciferase, Reporter Assay

24) Product Images from "Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch"

Article Title: Herpes simplex virus UL56 interacts with and regulates the Nedd4-family ubiquitin ligase Itch

Journal: Virology Journal

doi: 10.1186/1743-422X-7-179

Effects of UL56 and other viral proteins with a PY motif on Itch . (A) Itch is markedly decreased in cells stably expressing UL56 (Vero-GFP-UL56). Lysates from Vero, Vero-GFP-UL56, or Vero-GFP cells were analyzed for Itch and other Nedd4-family ubiquitin ligases. Itch was markedly decreased in Vero-GFP-UL56 cells. (B) VP5 did not decrease endogenous Itch. Vero cells were transfected with plasmids encoding VP5 (pcDNA-VP5) or control plasmids (pcDNA3.1(+)). The levels of Itch did not change in cells transfected with pcDNA-VP5. (C) VP5 and VP16, but not ICP0, caused the decrease of exogenous Itch. Vero cells were co-transfected with plasmids encoding Itch (pcDNA-Itch) and plasmids encoding a viral protein (pMyc-ICP0, pcDNA-VP5, or pcDNA-VP16) or control plasmids (pCMV-Myc or pcDNA3.1(+)). The levels of Itch decreased in cells transfected with pcDNA-VP5 or pcDNA-VP16. (D) Overexpression of Itch has no effect on the protein levels of VP5, VP16, or ICP0. Vero cells were co-transfected with plasmids encoding a viral protein (pMyc-ICP0, pcDNA-VP5 or pcDNA-VP16) and either pcDNA-Itch or control plasmids (pcDNA-3.1(+)). The levels of viral proteins did not change with the overexpression of Itch. β-actin was used as a loading control.
Figure Legend Snippet: Effects of UL56 and other viral proteins with a PY motif on Itch . (A) Itch is markedly decreased in cells stably expressing UL56 (Vero-GFP-UL56). Lysates from Vero, Vero-GFP-UL56, or Vero-GFP cells were analyzed for Itch and other Nedd4-family ubiquitin ligases. Itch was markedly decreased in Vero-GFP-UL56 cells. (B) VP5 did not decrease endogenous Itch. Vero cells were transfected with plasmids encoding VP5 (pcDNA-VP5) or control plasmids (pcDNA3.1(+)). The levels of Itch did not change in cells transfected with pcDNA-VP5. (C) VP5 and VP16, but not ICP0, caused the decrease of exogenous Itch. Vero cells were co-transfected with plasmids encoding Itch (pcDNA-Itch) and plasmids encoding a viral protein (pMyc-ICP0, pcDNA-VP5, or pcDNA-VP16) or control plasmids (pCMV-Myc or pcDNA3.1(+)). The levels of Itch decreased in cells transfected with pcDNA-VP5 or pcDNA-VP16. (D) Overexpression of Itch has no effect on the protein levels of VP5, VP16, or ICP0. Vero cells were co-transfected with plasmids encoding a viral protein (pMyc-ICP0, pcDNA-VP5 or pcDNA-VP16) and either pcDNA-Itch or control plasmids (pcDNA-3.1(+)). The levels of viral proteins did not change with the overexpression of Itch. β-actin was used as a loading control.

Techniques Used: Stable Transfection, Expressing, Transfection, Over Expression

25) Product Images from "High Guanine and Cytosine Content Increases mRNA Levels in Mammalian CellsMore GC Means More RNA"

Article Title: High Guanine and Cytosine Content Increases mRNA Levels in Mammalian CellsMore GC Means More RNA

Journal: PLoS Biology

doi: 10.1371/journal.pbio.0040180

Expression of GFP and IL2 Variants in Stably Transfected MCF-7 Cells MCF-7 cells were stably transfected with expression plasmids containing GFP (GC3 = 35%), EGFP (GC3 = 96%), wIL2 (GC3 = 7%), IL2 (GC3 = 41%), IL2-eIL2 (GC3 = 70%), eIL2 (GC3 = 100%), or with an empty pcDNA3.1 plasmid. The expression plasmids contained CMV promoters and were integrated in random genomic locations. Protein and mRNA was quantified in three to five individual clones for each transgene. (A) Flow cytometry measurements of GFP and EGFP protein levels. (B) Real-time RT-PCR measurements of GFP and EGFP mRNA levels. (C) ELISA measurements of the IL2 protein levels. (D) real-time RT-PCR measurements of IL2 mRNA levels. GFP and IL2 mRNA levels were normalized to GAPDH mRNA. The controls represent pcDNA3.1-transfected cells. The vertical axis in each graph represents arbitrary units.
Figure Legend Snippet: Expression of GFP and IL2 Variants in Stably Transfected MCF-7 Cells MCF-7 cells were stably transfected with expression plasmids containing GFP (GC3 = 35%), EGFP (GC3 = 96%), wIL2 (GC3 = 7%), IL2 (GC3 = 41%), IL2-eIL2 (GC3 = 70%), eIL2 (GC3 = 100%), or with an empty pcDNA3.1 plasmid. The expression plasmids contained CMV promoters and were integrated in random genomic locations. Protein and mRNA was quantified in three to five individual clones for each transgene. (A) Flow cytometry measurements of GFP and EGFP protein levels. (B) Real-time RT-PCR measurements of GFP and EGFP mRNA levels. (C) ELISA measurements of the IL2 protein levels. (D) real-time RT-PCR measurements of IL2 mRNA levels. GFP and IL2 mRNA levels were normalized to GAPDH mRNA. The controls represent pcDNA3.1-transfected cells. The vertical axis in each graph represents arbitrary units.

Techniques Used: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Clone Assay, Flow Cytometry, Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

26) Product Images from "Molecular cloning and characterization of a lipid phosphohydrolase that degrades sphingosine-1- phosphate and induces cell death"

Article Title: Molecular cloning and characterization of a lipid phosphohydrolase that degrades sphingosine-1- phosphate and induces cell death

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

doi:

Expression of mSPP1 increases ceramide levels and enhances apoptosis of NIH 3T3 fibroblasts. ( A ) NIH 3T3 fibroblasts were transfected with either pcDNA3.1 (open bars) or pcDNA3.1-mSPP1 (filled bars). Twenty-four hours after transfection, the medium was replaced with serum-free DMEM and sphingolipid metabolite levels were determined as described in Materials and Methods . ( B and C ) NIH 3T3 fibroblasts were transfected with pCEFL-GFP and either pcDNA3.1 (open bars) or pcDNA3.1-mSPP1 (filled bars). Twenty-four hours after transfection, the medium was replaced with serum-free DMEM (SF) in the absence or presence of C 2 -ceramide (CER, 7.5 μM), H 2 O 2 (100 μM), doxorubicin (DOXO, 0.2 μg/ml), staurosporine (Staur, 25 nM), 10% calf serum (CS), SPP (5 μM), or platelet-derived growth factor (10 ng/ml). After an additional 24 h ( B ) or 48 h ( C ), total GFP-expressing cells and GFP-expressing cells displaying rounded morphology were counted and percent apoptotic cells expressing GFP was determined as described in Materials and Methods . A minimum of 500 cells in each field were scored. Data are mean ± SE of three independent experiments, each one done in triplicate. ( D ) Each set of panels shows the same field from vector-transfected and mSPP1-expressing cells cultured in serum-free medium examined by fluorescence microscopy to visualize GFP (green, Lower ) and also stained with Hoechst dye (blue, Upper ) to visualize fragmented nuclei indicative of apoptosis. Note the typical condensed fragmented nuclei of apoptotic cells in mSPP1-transfected but not in vector-transfected cells after serum deprivation for 48 h, which correlates with the rounded cell morphology revealed by green fluorescence.
Figure Legend Snippet: Expression of mSPP1 increases ceramide levels and enhances apoptosis of NIH 3T3 fibroblasts. ( A ) NIH 3T3 fibroblasts were transfected with either pcDNA3.1 (open bars) or pcDNA3.1-mSPP1 (filled bars). Twenty-four hours after transfection, the medium was replaced with serum-free DMEM and sphingolipid metabolite levels were determined as described in Materials and Methods . ( B and C ) NIH 3T3 fibroblasts were transfected with pCEFL-GFP and either pcDNA3.1 (open bars) or pcDNA3.1-mSPP1 (filled bars). Twenty-four hours after transfection, the medium was replaced with serum-free DMEM (SF) in the absence or presence of C 2 -ceramide (CER, 7.5 μM), H 2 O 2 (100 μM), doxorubicin (DOXO, 0.2 μg/ml), staurosporine (Staur, 25 nM), 10% calf serum (CS), SPP (5 μM), or platelet-derived growth factor (10 ng/ml). After an additional 24 h ( B ) or 48 h ( C ), total GFP-expressing cells and GFP-expressing cells displaying rounded morphology were counted and percent apoptotic cells expressing GFP was determined as described in Materials and Methods . A minimum of 500 cells in each field were scored. Data are mean ± SE of three independent experiments, each one done in triplicate. ( D ) Each set of panels shows the same field from vector-transfected and mSPP1-expressing cells cultured in serum-free medium examined by fluorescence microscopy to visualize GFP (green, Lower ) and also stained with Hoechst dye (blue, Upper ) to visualize fragmented nuclei indicative of apoptosis. Note the typical condensed fragmented nuclei of apoptotic cells in mSPP1-transfected but not in vector-transfected cells after serum deprivation for 48 h, which correlates with the rounded cell morphology revealed by green fluorescence.

Techniques Used: Expressing, Transfection, Derivative Assay, Plasmid Preparation, Cell Culture, Fluorescence, Microscopy, Staining

Characterization of mSPP1 activity in transfected cells. ( A ) Phosphohydrolase activity in cytosolic and membrane fractions. HEK293 cells were transfected with pcDNA3.1 or pcDNA3.1-mSPP1. Cytosolic (open bars) and membrane fractions (filled bars) were isolated, and SPP phosphohydrolase activity was determined by measuring hydrolysis of [ 32 P]SPP as described in Materials and Methods . Results are means ± SD of triplicate determinations. Similar results were obtained in three additional experiments. ( Inset ) Phosphohydrolase activity of mSPP1 immunoprecipitated with anti-c-myc. Myc-tagged mSPP1 was partially purified from HEK293 cells transfected with empty vector or myc-tagged mSPP1 as described in Materials and Methods , and phosphohydrolase activity was measured with 32 P-SPP (open bars) or 32 P-LPA (filled bars). Data are expressed as nmol hydrolyzed per 30 min. ( B ) Inhibition of SPP phosphohydrolase activity by various lipids. Membrane fractions from HEK 293 fibroblasts transfected with pcDNA3.1-mSPP1 were incubated with [ 32 P]SPP (10 μM) for 30 min at 37°C in the absence or presence of the indicated lipids (100 μM), and phosphohydrolase activity was measured. Data are expressed as percent inhibition and are means ± SD. Dihydro-Sph, sphinganine; Sph, sphingosine; Dihydro-SPP, sphinganine-1-phosphate; C-8 C-1-P, C 8 -ceramide-1-phosphate; SPC, sphingosylphosphocholine; LPA, lysophosphatidic acid; PA, phosphatidic acid; G3P, glycerol-3-phosphate. ( C ) mSPP1 specifically dephosphorylates SPP. Phosphohydrolase activity was measured with 32 P-labeled lipids: SPP, LPA, PA, and ceramide-1-phosphate (CER-1-P). Results are expressed as fold stimulation of phosphohydrolase activity compared with vector-transfected HEK293 cells.
Figure Legend Snippet: Characterization of mSPP1 activity in transfected cells. ( A ) Phosphohydrolase activity in cytosolic and membrane fractions. HEK293 cells were transfected with pcDNA3.1 or pcDNA3.1-mSPP1. Cytosolic (open bars) and membrane fractions (filled bars) were isolated, and SPP phosphohydrolase activity was determined by measuring hydrolysis of [ 32 P]SPP as described in Materials and Methods . Results are means ± SD of triplicate determinations. Similar results were obtained in three additional experiments. ( Inset ) Phosphohydrolase activity of mSPP1 immunoprecipitated with anti-c-myc. Myc-tagged mSPP1 was partially purified from HEK293 cells transfected with empty vector or myc-tagged mSPP1 as described in Materials and Methods , and phosphohydrolase activity was measured with 32 P-SPP (open bars) or 32 P-LPA (filled bars). Data are expressed as nmol hydrolyzed per 30 min. ( B ) Inhibition of SPP phosphohydrolase activity by various lipids. Membrane fractions from HEK 293 fibroblasts transfected with pcDNA3.1-mSPP1 were incubated with [ 32 P]SPP (10 μM) for 30 min at 37°C in the absence or presence of the indicated lipids (100 μM), and phosphohydrolase activity was measured. Data are expressed as percent inhibition and are means ± SD. Dihydro-Sph, sphinganine; Sph, sphingosine; Dihydro-SPP, sphinganine-1-phosphate; C-8 C-1-P, C 8 -ceramide-1-phosphate; SPC, sphingosylphosphocholine; LPA, lysophosphatidic acid; PA, phosphatidic acid; G3P, glycerol-3-phosphate. ( C ) mSPP1 specifically dephosphorylates SPP. Phosphohydrolase activity was measured with 32 P-labeled lipids: SPP, LPA, PA, and ceramide-1-phosphate (CER-1-P). Results are expressed as fold stimulation of phosphohydrolase activity compared with vector-transfected HEK293 cells.

Techniques Used: Activity Assay, Transfection, Isolation, Immunoprecipitation, Purification, Plasmid Preparation, Inhibition, Incubation, Labeling

27) Product Images from "The effects inhibiting the proliferation of cancer cells by far-infrared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A"

Article Title: The effects inhibiting the proliferation of cancer cells by far-infrared radiation (FIR) are controlled by the basal expression level of heat shock protein (HSP) 70A

Journal: Medical Oncology (Northwood, London, England)

doi: 10.1007/s12032-007-9020-4

Over expression of HSP70 prevents the suppression of cell growth and induction of cell hypertrophy by FIR. ( a ) Real-time RT-PCR of HSP70 expression. Cells overexpressing HSP70 were established. ( b ) Representative Western blots demonstrating increased expression of HSP70 in stably transfected A431 and HSC3 cells. Lane 1, wild-type cells; lane 2, cells transfected with empty pcDNA3.1(+); lane 3, cells transfected pcDNA3.1-HSP70A. ( c ) Cell proliferation of empty vector-transfected and HSP70-overexpressing cells. Cell proliferation was the same in FIR-irradiated and untreated A431 cells overexpressing HSP70. In contrast, in HSC3 cells, over expression of HSP70 prevented FIR inhibition of cell proliferation. ( d ) BrdU incorporation in wild-type, empty vector-transfected, and HSP70-overexpressing cells treated with FIR. ( e ) Hematoxylin and eosin staining of wild-type, empty vector-transfected, and HSP70A-overexpressing A431 and HSC3 cells after 5 days of transfection. Note that the morphology of HSP70A-transfected HSC3 cells was unaffected by FIR irradiation
Figure Legend Snippet: Over expression of HSP70 prevents the suppression of cell growth and induction of cell hypertrophy by FIR. ( a ) Real-time RT-PCR of HSP70 expression. Cells overexpressing HSP70 were established. ( b ) Representative Western blots demonstrating increased expression of HSP70 in stably transfected A431 and HSC3 cells. Lane 1, wild-type cells; lane 2, cells transfected with empty pcDNA3.1(+); lane 3, cells transfected pcDNA3.1-HSP70A. ( c ) Cell proliferation of empty vector-transfected and HSP70-overexpressing cells. Cell proliferation was the same in FIR-irradiated and untreated A431 cells overexpressing HSP70. In contrast, in HSC3 cells, over expression of HSP70 prevented FIR inhibition of cell proliferation. ( d ) BrdU incorporation in wild-type, empty vector-transfected, and HSP70-overexpressing cells treated with FIR. ( e ) Hematoxylin and eosin staining of wild-type, empty vector-transfected, and HSP70A-overexpressing A431 and HSC3 cells after 5 days of transfection. Note that the morphology of HSP70A-transfected HSC3 cells was unaffected by FIR irradiation

Techniques Used: Over Expression, Quantitative RT-PCR, Expressing, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Irradiation, Inhibition, BrdU Incorporation Assay, Staining

28) Product Images from "Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy"

Article Title: Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.46.6.1896-1905.2002

Sequence analysis of env clones derived from plasma virus. Prior to (d0) and 14 days after (d14) treatment with T-20, the full-length env gene was amplified from viral RNA by RT-PCR and cloned into pCDNA3.1. The sequences of multiple clones derived from subjects 30-1 (A) and 30-3 (B) were analyzed using Sequencher software and aligned against the LAI consensus sequence. The sequence of the entire HR1 domain is illustrated. The vertical shaded rectangles highlight the GIV motif.
Figure Legend Snippet: Sequence analysis of env clones derived from plasma virus. Prior to (d0) and 14 days after (d14) treatment with T-20, the full-length env gene was amplified from viral RNA by RT-PCR and cloned into pCDNA3.1. The sequences of multiple clones derived from subjects 30-1 (A) and 30-3 (B) were analyzed using Sequencher software and aligned against the LAI consensus sequence. The sequence of the entire HR1 domain is illustrated. The vertical shaded rectangles highlight the GIV motif.

Techniques Used: Sequencing, Clone Assay, Derivative Assay, Amplification, Reverse Transcription Polymerase Chain Reaction, Software

29) Product Images from "Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production"

Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109640

SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.
Figure Legend Snippet: SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

Techniques Used: Transfection, Immunoprecipitation, SDS Page

SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.
Figure Legend Snippet: SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

Techniques Used: Purification, Transfection, Produced

SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.
Figure Legend Snippet: SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Standard Deviation, Activity Assay, Luciferase, Protein Concentration

Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.
Figure Legend Snippet: Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

Techniques Used: Transfection

SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.
Figure Legend Snippet: SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

Techniques Used: Transfection, Standard Deviation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

30) Product Images from "Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production"

Article Title: Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

Journal: PLoS ONE

doi: 10.1371/journal.pone.0109640

SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.
Figure Legend Snippet: SOCS1 physically interacts with RhTRIM5α. HEK293T cells were co-transfected with 1.0 µg of pRhTRIM5α-HA and 2.0 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 3.0 µg per sample with pcDNA3.1. Two days post-transfection, whole cell lysates were subjected to immunoprecipitation for RhTRIM5α with anti-HA antibody. Input lysates (left panels) and precipitated proteins (right panels) were separated by SDS-PAGE and proteins were detected by immunoblot analyses with anti-SOCS1 (upper panels) and anti-HA (RhTRIM5α-HA, lower panels) antibodies.

Techniques Used: Transfection, Immunoprecipitation, SDS Page

SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.
Figure Legend Snippet: SOCS1 is detected in purified HIV-1 VLPs. HEK293T cells were co-transfected with 2.4 µg of pNL4-3, 2.4 µg of pRhTRIM5α-HA and 2.4 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 7.2 µg per sample with pcDNA3.1. (A) Relative viral titer in the supernatants was analyzed with TZM-bl indicator cells two days post-transfection. The titer of virus produced in the absence of RhTRIM5α and SOCS1 (Ct) was arbitrarily set as 100%. S1, T5α or T5α/S1 indicate virions produced in the presence of SOCS1 alone, RhTRIM5α-HA alone or both RhTRIM5α-HA and SOCS1, respectively. Producer cell lysates (B, left panels) and purified HIV-1 VLPs purified through a 20% sucrose layer (B, right panels) were subjected to immunoblot analysis. Proteins were detected with anti-HA (RhTRIM5α-HA, top panels), anti-SOCS1 (middle panels) and anti-HIV-1 p24 (bottom panels) antibodies.

Techniques Used: Purification, Transfection, Produced

SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.
Figure Legend Snippet: SOCS1 affects RhTRIM5α expression in a dose-dependent manner. HEK293T cells were co-transfected with 0.1 µg of pNL4-3 or pUC18 with increasing amounts of pHuSOCS1 (0, 0.0375, 0.075, 0.15, 0.3 and 0.6 µg) and with or without 0.3 µg of pRhTRIM5α-HA. The amount of plasmid DNA was adjusted to 1.0 µg with pcDNA3.1. Two days post-transfection, viral titer in the culture supernatants was analyzed with TZM-bl indicator cells. (A) Relative viral titer obtained without exogenous SOCS1 and RhTRIM5α was arbitrarily set as 100%. The results are shown as an average of three independent experiments with standard deviation. (B) Whole cell lysates in panel (A) were subjected to immunoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (C) Relative band intensities of RhTRIM5α and SOCS1 in panel (B) were normalized with that of GAPDH. The normalized band intensity of RhTRIM5α (upper panels) in the absence of SOCS1 and that of SOCS1 (lower panels) without exogenous SOCS1 were arbitrarily set as 100%. (D) Effect of SOCS1 expression on transcriptional activity. HEK293T cell were co-transfected with 0.05 µg of pGL4.84-EF1α-hRlucCP (EF1α) or pcDNA3.1-Luc (CMV) together with 0.45 µg of pcDNA3.1 or pHuSOCS1. The amount of plasmid DNA was adjusted to 0.5 µg with pcDNA3.1. Two days after transfection, luciferase activity was determined and normalized with protein concentration (Luc/protein). The results are shown as an average of three independent experiments with standard deviation.

Techniques Used: Expressing, Transfection, Plasmid Preparation, Standard Deviation, Activity Assay, Luciferase, Protein Concentration

Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.
Figure Legend Snippet: Reduction of RhTRIM5α by SOCS1 is proteasome-independent. HEK293T cells were transfected with 0.1 µg of pNL4-3, 0.3 µg of pRhTRIM5α-HA with or without 0.6 µg of pHuSOCS1. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Twenty-four hours after transfection, cells were treated with 30 µM of MG115 (lanes 3 and 4) or 30 µM of MG132 (lanes 5 and 6) for 16 hours. Whole cell lysates were subjected to immunoblot analyses with anti-HA (RhTRIM5α-HA, top panel), anti-SOCS1 (middle panel), anti-IκBα and anti-GAPDH (as loading control, bottom panel) antibodies.

Techniques Used: Transfection

SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.
Figure Legend Snippet: SOCS1 reverses RhTRIM5α-mediated late restriction of HIV-1 replication. (A) HEK293T cells were transfected with 0.1 µg of pNL4-3 and with or without 0.6 µg of pHuSOCS1 (S1) and/or 0.3 µg of pRhTRIM5α-HA (T5α). Ct represents transfection with the control vectors. T5α/S1 indicates that S1 and T5α were co-transfected besides pNL4-3. The total amount of plasmids transfected was adjusted to 1.0 µg per sample with pcDNA3.1. Two days post-transfection, relative viral titer in the supernatants was analyzed with TZM-bl indicator cells. Average of results from four independent experiments is shown with standard deviation. (B) The amount of p24 antigen in the supernatants was quantified with p24-specific ELISA. Data were obtained from the same experimental sets shown in panel A. (C) Twenty-four μg of whole cell lysates were subjected to immnoblot analyses with anti-HIV-1 p24, anti-HA (RhTRIM5α-HA), anti-SOCS1 and anti-GAPDH antibodies. (D) Relative RhTRIM5α protein expression level was determined by densitometry analysis in panel (C). The band intensity for T5α was arbitrarily set as 100%. (E) Relative Rh trim5α mRNA expression determined by quantitative RT-PCR. The value for T5α was arbitrarily set as 100%. The results are shown as an average obtained in four independent experiments with standard deviation.

Techniques Used: Transfection, Standard Deviation, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

31) Product Images from ""

Article Title:

Journal: Molecular Biology of the Cell

doi: 10.1091/mbc.E06-12-1064

Cx43 regulates TGF-β signaling. (A) Effect of Cx depletion on transcriptional activation of the TGF-β–targeted gene. HL-1 cells were cotransfected with the reporter vector p3TP-lux and internal control plasmid pRL-SV together with pDECAP empty vector, pDECAP vector expressing N-terminal Cx43 dsRNA (pDECAP.Cx43.NT), C-terminal Cx43 dsRNA (pDECAP.Cx43.CT), N-terminal Cx40 dsRNA (pDECAP.Cx40.NT), C-terminal Cx40 dsRNA (pDECAP.Cx40.CT), N-terminal Cx45 dsRNA (pDECAP.Cx45.NT), or C-terminal Cx45 dsRNA (pDECAP.Cx45.CT). (B) Cx43 is specifically required for nuclear translocation of Smad2/3 and Smad4 in cardiomyocytes. HL-1 cells were transfected with pDECAP.Cx43.CT, pDECAP.Cx43.NT, and pDECAP.Cx40.CT together with a transfectant indicator, pcDNA.3.1.mRFP1. Cells were fixed and stained with anti-Smad2/3 (left) or anti-Smad4 (right) monoclonal antibodies followed by Alexa Fluor 488 goat anti-mouse IgG and were examined by confocal fluorescence microscopy. (C) The TD of Cx43 is required for Smad-induced transcriptional activity. HL-1 cells (left) were transfected with the reporter vector p3TP-lux and internal control plasmid pRL-SV together with pcDNA3.1.Cx43, pcDNA3.1.Cx43ΔTD, or pcDNA3.1.Cx43JM228-263. HeLa cells (right) were transfected with the reporter vector CAGA 12 -luc, internal control plasmid pRL-CMV, and together with pcDNA3.1.Cx43, pcDNA3.1.Cx43ΔTD, or pcDNA3.1.Cx43JM228-263. All samples were harvested together at the 40-h time point, and luciferase activity was measured and normalized. The data shown in A and C are means and SDs (error bars) of a representative experiment performed in triplicate and were evaluated for statistical significance with two-tailed Student's test. *p
Figure Legend Snippet: Cx43 regulates TGF-β signaling. (A) Effect of Cx depletion on transcriptional activation of the TGF-β–targeted gene. HL-1 cells were cotransfected with the reporter vector p3TP-lux and internal control plasmid pRL-SV together with pDECAP empty vector, pDECAP vector expressing N-terminal Cx43 dsRNA (pDECAP.Cx43.NT), C-terminal Cx43 dsRNA (pDECAP.Cx43.CT), N-terminal Cx40 dsRNA (pDECAP.Cx40.NT), C-terminal Cx40 dsRNA (pDECAP.Cx40.CT), N-terminal Cx45 dsRNA (pDECAP.Cx45.NT), or C-terminal Cx45 dsRNA (pDECAP.Cx45.CT). (B) Cx43 is specifically required for nuclear translocation of Smad2/3 and Smad4 in cardiomyocytes. HL-1 cells were transfected with pDECAP.Cx43.CT, pDECAP.Cx43.NT, and pDECAP.Cx40.CT together with a transfectant indicator, pcDNA.3.1.mRFP1. Cells were fixed and stained with anti-Smad2/3 (left) or anti-Smad4 (right) monoclonal antibodies followed by Alexa Fluor 488 goat anti-mouse IgG and were examined by confocal fluorescence microscopy. (C) The TD of Cx43 is required for Smad-induced transcriptional activity. HL-1 cells (left) were transfected with the reporter vector p3TP-lux and internal control plasmid pRL-SV together with pcDNA3.1.Cx43, pcDNA3.1.Cx43ΔTD, or pcDNA3.1.Cx43JM228-263. HeLa cells (right) were transfected with the reporter vector CAGA 12 -luc, internal control plasmid pRL-CMV, and together with pcDNA3.1.Cx43, pcDNA3.1.Cx43ΔTD, or pcDNA3.1.Cx43JM228-263. All samples were harvested together at the 40-h time point, and luciferase activity was measured and normalized. The data shown in A and C are means and SDs (error bars) of a representative experiment performed in triplicate and were evaluated for statistical significance with two-tailed Student's test. *p

Techniques Used: Activation Assay, Plasmid Preparation, Expressing, Translocation Assay, Transfection, Staining, Fluorescence, Microscopy, Activity Assay, Luciferase, Two Tailed Test

Cx43 cooperatively interacts with TGF-β in regulating TGF-β/Smad signaling. (A and B) Cx43 depletion abrogates TGF-β–induced Smad-mediated transcription. HL-1 (A) or HepG2 (B) cells were transfected with p3TP-lux and pRL-SV and together with pDECAP.Cx43.NT, pDECAP.Cx43.CT, or pDECAP.Cx40.CT, which served as a negative control. (C) Cx43 cooperatively potentiates TGF-β–induced transcriptional activity. HepG2 cells transfected with p3TP-lux, pRL-SV and a dose of pcDNA.3.1.Cx43. After transfection, cells were incubated for 32 h in 10% FBS/Claycomb medium or FBS/DMEM, followed by inductions for 8 h with or without 5 ng/ml TGF-β1 in A–C. (D) Activated TGF-β receptor TβRI/ALK5 is required for Cx43-mediated TGF-β/Smad activity in HL-1 cardiomyocytes. HL-1 cells were transfected with internal control plasmid pRL-SV and the indicated dose of the reporter vector CAGA 12 -luc (left) or p3TP-lux (right). (E) TβRI/ALK5 kinase activity is required for Cx43-mediated TGF-β/Smad activity in HeLa cells. HeLa cells were transfected with the reporter vector CAGA 12 -luc, internal control plasmid pRL-CMV, and the indicated dose of pcDNA3.1.Cx43. Immediately after transfection, cells were incubated for 40 h in 10% FBS/DMEM containing DMSO alone or 10 μM SB-431542 in D and E. For luciferase assays, all samples were harvested together at the 40-h time point after transfection and luciferase activity was then measured and normalized. All data shown in A–E are means and SDs (error bars) of a representative experiment performed in triplicate.
Figure Legend Snippet: Cx43 cooperatively interacts with TGF-β in regulating TGF-β/Smad signaling. (A and B) Cx43 depletion abrogates TGF-β–induced Smad-mediated transcription. HL-1 (A) or HepG2 (B) cells were transfected with p3TP-lux and pRL-SV and together with pDECAP.Cx43.NT, pDECAP.Cx43.CT, or pDECAP.Cx40.CT, which served as a negative control. (C) Cx43 cooperatively potentiates TGF-β–induced transcriptional activity. HepG2 cells transfected with p3TP-lux, pRL-SV and a dose of pcDNA.3.1.Cx43. After transfection, cells were incubated for 32 h in 10% FBS/Claycomb medium or FBS/DMEM, followed by inductions for 8 h with or without 5 ng/ml TGF-β1 in A–C. (D) Activated TGF-β receptor TβRI/ALK5 is required for Cx43-mediated TGF-β/Smad activity in HL-1 cardiomyocytes. HL-1 cells were transfected with internal control plasmid pRL-SV and the indicated dose of the reporter vector CAGA 12 -luc (left) or p3TP-lux (right). (E) TβRI/ALK5 kinase activity is required for Cx43-mediated TGF-β/Smad activity in HeLa cells. HeLa cells were transfected with the reporter vector CAGA 12 -luc, internal control plasmid pRL-CMV, and the indicated dose of pcDNA3.1.Cx43. Immediately after transfection, cells were incubated for 40 h in 10% FBS/DMEM containing DMSO alone or 10 μM SB-431542 in D and E. For luciferase assays, all samples were harvested together at the 40-h time point after transfection and luciferase activity was then measured and normalized. All data shown in A–E are means and SDs (error bars) of a representative experiment performed in triplicate.

Techniques Used: Transfection, Negative Control, Activity Assay, Incubation, Plasmid Preparation, Luciferase

Cx43 induces Smad2 for nuclear translocation. Time-lapse observation of subcellular distribution of Smad2 in HeLa cells stably expressing GFPSmad2 were transfected with pcDNA3.1.Cx43 together with a transfectant indicator, pcDNA.3.1.mRFP1.
Figure Legend Snippet: Cx43 induces Smad2 for nuclear translocation. Time-lapse observation of subcellular distribution of Smad2 in HeLa cells stably expressing GFPSmad2 were transfected with pcDNA3.1.Cx43 together with a transfectant indicator, pcDNA.3.1.mRFP1.

Techniques Used: Translocation Assay, Stable Transfection, Expressing, Transfection

32) Product Images from "Herpes Simplex Virus Type 2 Triggers Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency and Collaborates with HIV-1 Tat"

Article Title: Herpes Simplex Virus Type 2 Triggers Reactivation of Kaposi's Sarcoma-Associated Herpesvirus from Latency and Collaborates with HIV-1 Tat

Journal: PLoS ONE

doi: 10.1371/journal.pone.0031652

HIV-1 Tat cooperates with HSV-2 to induce KSHV lytic cycle replication. ( A ). Effect of Tat on ORF26 mRNA expression in HSV-2-infected BCBL-1 cells. Real-time quantitative PCR was performed to detect KSHV ORF26 mRNA expression in Mock-treated and pcDNA3.1-transfected BCBL-1 cells ( Mock+pcDNA ), Mock-treated and pTat-transfected BCBL-1 cells ( Mock+Tat ), or HSV-2-infected and pcDNA3.1-transfected BCBL-1 cells ( HSV-2+pcDNA ), HSV-2-infected and pTat-transfected BCBL-1 cells ( HSV-2+Tat ) for 6, 24, 48 and 72 hours. The results shown were from three independent experiments performed in triplicate. ** p
Figure Legend Snippet: HIV-1 Tat cooperates with HSV-2 to induce KSHV lytic cycle replication. ( A ). Effect of Tat on ORF26 mRNA expression in HSV-2-infected BCBL-1 cells. Real-time quantitative PCR was performed to detect KSHV ORF26 mRNA expression in Mock-treated and pcDNA3.1-transfected BCBL-1 cells ( Mock+pcDNA ), Mock-treated and pTat-transfected BCBL-1 cells ( Mock+Tat ), or HSV-2-infected and pcDNA3.1-transfected BCBL-1 cells ( HSV-2+pcDNA ), HSV-2-infected and pTat-transfected BCBL-1 cells ( HSV-2+Tat ) for 6, 24, 48 and 72 hours. The results shown were from three independent experiments performed in triplicate. ** p

Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Transfection

33) Product Images from "Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis *"

Article Title: Porcine circovirus type 2 capsid protein induces unfolded protein response with subsequent activation of apoptosis *

Journal: Journal of Zhejiang University. Science. B

doi: 10.1631/jzus.B1600208

Rep and Cap, but not ORF3, induced unfolded protein response via PERK activation PK-15 cells were transfected with the indicated plasmids (p-Flag stands for pcDNA3.1-Flag, p-EGFP for pcDNA3.1-EGFP, p-ORF3-EGFP for pcDNA3.1-ORF3-EGFP, p-Rep for p-Rep-Flag, p-Cap and p-Cap-Flag, the same for the following figures) for 12, 24, 36, and 48 h. Western blotting was performed to visualize phosphorylated forms of PERK (p-PERK) and eIF2α (p-eIF2α), total PERK (t-PERK) and total eIF2α (t-eIF2α), and ER stress marker GRP78 in lysates of cells expressing Rep or Cap (a) and of those expressing ORF3 (b). Expressions of the fusion proteins Cap, Rep, and ORF3 were revealed by antibodies to Flag or EGFP
Figure Legend Snippet: Rep and Cap, but not ORF3, induced unfolded protein response via PERK activation PK-15 cells were transfected with the indicated plasmids (p-Flag stands for pcDNA3.1-Flag, p-EGFP for pcDNA3.1-EGFP, p-ORF3-EGFP for pcDNA3.1-ORF3-EGFP, p-Rep for p-Rep-Flag, p-Cap and p-Cap-Flag, the same for the following figures) for 12, 24, 36, and 48 h. Western blotting was performed to visualize phosphorylated forms of PERK (p-PERK) and eIF2α (p-eIF2α), total PERK (t-PERK) and total eIF2α (t-eIF2α), and ER stress marker GRP78 in lysates of cells expressing Rep or Cap (a) and of those expressing ORF3 (b). Expressions of the fusion proteins Cap, Rep, and ORF3 were revealed by antibodies to Flag or EGFP

Techniques Used: Activation Assay, Transfection, Western Blot, Marker, Expressing

34) Product Images from "Cloning and Functional Characterization of Human SMCT2 (SLC5A12) and Expression Pattern of the Transporter in Kidney"

Article Title: Cloning and Functional Characterization of Human SMCT2 (SLC5A12) and Expression Pattern of the Transporter in Kidney

Journal: Biochimica et biophysica acta

doi: 10.1016/j.bbamem.2007.06.031

Dose-response relationship for the inhibition of human SMCT2-mediated [ 14 C]nicotinate uptake by unlabeled nicotinate, lactate, and butyrate (A) and inhibition of human SMCT2-mediated nicotinate uptake by NSAIDs (B). Human SMCT2 cDNA was expressed in HRPE cells. Cells transfected with pcDNA3.1 vector served to determine endogenous transport. Uptake of [ 14 C]nicotinate (30 μM) was measured in Na + -containing buffer either in the presence of increasing concentrations of unlabelled nicotinate, L-lactate, or butyrate (A) or various NSAIDs (200 μM) (B). Uptake measured in cells transfected with vector alone was subtracted from corresponding uptake measured in cells transfected with cDNA to calculate cDNA-specific uptake. Results are expressed as percentage of control uptake (100 %) measured in the absence of inhibitors. The degree of statistical significance for nicotinate uptake measured in the presence of NSAIDs compared to nicotinate uptake measured in the absence of various NSAIDs in hSMCT2 cDNA-transfected cells is indicated by * P
Figure Legend Snippet: Dose-response relationship for the inhibition of human SMCT2-mediated [ 14 C]nicotinate uptake by unlabeled nicotinate, lactate, and butyrate (A) and inhibition of human SMCT2-mediated nicotinate uptake by NSAIDs (B). Human SMCT2 cDNA was expressed in HRPE cells. Cells transfected with pcDNA3.1 vector served to determine endogenous transport. Uptake of [ 14 C]nicotinate (30 μM) was measured in Na + -containing buffer either in the presence of increasing concentrations of unlabelled nicotinate, L-lactate, or butyrate (A) or various NSAIDs (200 μM) (B). Uptake measured in cells transfected with vector alone was subtracted from corresponding uptake measured in cells transfected with cDNA to calculate cDNA-specific uptake. Results are expressed as percentage of control uptake (100 %) measured in the absence of inhibitors. The degree of statistical significance for nicotinate uptake measured in the presence of NSAIDs compared to nicotinate uptake measured in the absence of various NSAIDs in hSMCT2 cDNA-transfected cells is indicated by * P

Techniques Used: Inhibition, Transfection, Plasmid Preparation

Functional expression of human SMCT2 (SLC5A12) in HRPE cells. HRPE cells were transfected with either pcDNA3.1 vector alone (open bars) or human SMCT2 cDNA (closed bars). ( A, B ) Uptake of [ 14 C]lactate (500 μM), [ 14 C]pyruvate (500 μM), and [ 14 C]nicotinate (30 μM) was measured in the presence of Na + . ( C ) Uptake of [ 14 C]nicotinate was measured either in control buffer (25 mM Hepes/Tris, pH 7.5, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose, and 140 mM NaCl) or in buffer in which NaCl was replaced with 140 mM of KCl or LiCl. Where indicated, uptake measured in cDNA-transfected cells was significantly different from the corresponding uptake measured in vector-transfected cells (* P
Figure Legend Snippet: Functional expression of human SMCT2 (SLC5A12) in HRPE cells. HRPE cells were transfected with either pcDNA3.1 vector alone (open bars) or human SMCT2 cDNA (closed bars). ( A, B ) Uptake of [ 14 C]lactate (500 μM), [ 14 C]pyruvate (500 μM), and [ 14 C]nicotinate (30 μM) was measured in the presence of Na + . ( C ) Uptake of [ 14 C]nicotinate was measured either in control buffer (25 mM Hepes/Tris, pH 7.5, 5.4 mM KCl, 1.8 mM CaCl 2 , 0.8 mM MgSO 4 , 5 mM glucose, and 140 mM NaCl) or in buffer in which NaCl was replaced with 140 mM of KCl or LiCl. Where indicated, uptake measured in cDNA-transfected cells was significantly different from the corresponding uptake measured in vector-transfected cells (* P

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

35) Product Images from "Kaiso protects human umbilical vein endothelial cells against apoptosis by differentially regulating the expression of B-cell CLL/lymphoma 2 family members"

Article Title: Kaiso protects human umbilical vein endothelial cells against apoptosis by differentially regulating the expression of B-cell CLL/lymphoma 2 family members

Journal: Scientific Reports

doi: 10.1038/s41598-017-07559-0

Expression of BCL2, BAX and BIK in Kaiso-modified endothelial cells. Cells were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA1 and were cultured at 37 °C for 48 h. The expression of BCL2, BAX and BIK was evaluated using quantitative real-time PCR ( A ) and Western blot ( B ). The untreated cells served as a blank. Tubulin served as a loading control. Values are presented as mean ± SD, **p
Figure Legend Snippet: Expression of BCL2, BAX and BIK in Kaiso-modified endothelial cells. Cells were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA1 and were cultured at 37 °C for 48 h. The expression of BCL2, BAX and BIK was evaluated using quantitative real-time PCR ( A ) and Western blot ( B ). The untreated cells served as a blank. Tubulin served as a loading control. Values are presented as mean ± SD, **p

Techniques Used: Expressing, Modification, Transfection, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot

Selective silencing and overexpression of Kaiso in endothelial cells. HUVECs ( A , B ) and HMEC-1s ( D , E ) were transfected with NC-siRNA or Kaiso-siRNA1 and silencing efficiency was evaluated by quantitative real-time PCR ( A and D ) and Western blot ( B and E ) at 48, 60 or 72 h post transfection. HUVECs ( C ) and HMEC-1s ( F ) were transfected with pCDNA3.1 or pCDNA3.1-Kaiso and Kaiso expression was evaluated by Western blot at 48 and 72 h after transfection. GAPDH served as a loading control. Values are presented as mean ± SD, **p
Figure Legend Snippet: Selective silencing and overexpression of Kaiso in endothelial cells. HUVECs ( A , B ) and HMEC-1s ( D , E ) were transfected with NC-siRNA or Kaiso-siRNA1 and silencing efficiency was evaluated by quantitative real-time PCR ( A and D ) and Western blot ( B and E ) at 48, 60 or 72 h post transfection. HUVECs ( C ) and HMEC-1s ( F ) were transfected with pCDNA3.1 or pCDNA3.1-Kaiso and Kaiso expression was evaluated by Western blot at 48 and 72 h after transfection. GAPDH served as a loading control. Values are presented as mean ± SD, **p

Techniques Used: Over Expression, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Expressing

P120ctn participates in the gene regulation of BCL2, BAX and BIK by Kaiso. HUVECs were transfected with pCDNA3.1-Kaiso for 48 h and stained with polyclonal rabbit anti-Kaiso (H-154) antibody (in red color) and monoclonal mouse anti-p120ctn antibody (in green color) ( A ). Cell nuclei were stained with DAPI ( A ,a). Arrowheads show the particle like structures where both Kaiso and p120ctn were observed ( A ,d). Scale bar represents 10 μm. HUVECs and HMEC-1s were co-transfected with pCMV-flag-Kaiso and pCMV-myc-p120ctn, and 48 h later the interaction of Kaiso and p120ctn was evaluated by co-immunoprecipitation using monoclonal mouse anti-FLAG antibody and monoclonal mouse anti-Myc tag antibody respectively ( B ). HUVECs were transfected with NC-siRNA, p120ctn-siRNA, pCDNA3.1 and pCDNA3.1-p120ctn respectively, and p120ctn expression was evaluated by Western blot 48 h after transfection ( C ). GAPDH served as a loading control. Values are presented as mean ± SD, *p
Figure Legend Snippet: P120ctn participates in the gene regulation of BCL2, BAX and BIK by Kaiso. HUVECs were transfected with pCDNA3.1-Kaiso for 48 h and stained with polyclonal rabbit anti-Kaiso (H-154) antibody (in red color) and monoclonal mouse anti-p120ctn antibody (in green color) ( A ). Cell nuclei were stained with DAPI ( A ,a). Arrowheads show the particle like structures where both Kaiso and p120ctn were observed ( A ,d). Scale bar represents 10 μm. HUVECs and HMEC-1s were co-transfected with pCMV-flag-Kaiso and pCMV-myc-p120ctn, and 48 h later the interaction of Kaiso and p120ctn was evaluated by co-immunoprecipitation using monoclonal mouse anti-FLAG antibody and monoclonal mouse anti-Myc tag antibody respectively ( B ). HUVECs were transfected with NC-siRNA, p120ctn-siRNA, pCDNA3.1 and pCDNA3.1-p120ctn respectively, and p120ctn expression was evaluated by Western blot 48 h after transfection ( C ). GAPDH served as a loading control. Values are presented as mean ± SD, *p

Techniques Used: Transfection, Staining, Immunoprecipitation, Expressing, Western Blot

KBSs take part in the Kaiso mediated transcriptional regulation of BCL2, BAX and BIK. HUVECs were transfected with recombinant firefly luciferase reporter constructs pGL3b-BCL2 ( B ), pGL4.1-BAX ( C ) or pGL4.1-BIK ( D ) together with pCDNA3.1 or pCDNA3.1-Kaiso. The KBS-mutated constructs were negative controls. Renilla luciferase reporter pRL-TK was an internal control. The untreated cells served as a blank. Relative luciferase activity is presented as mean ± SD. *p
Figure Legend Snippet: KBSs take part in the Kaiso mediated transcriptional regulation of BCL2, BAX and BIK. HUVECs were transfected with recombinant firefly luciferase reporter constructs pGL3b-BCL2 ( B ), pGL4.1-BAX ( C ) or pGL4.1-BIK ( D ) together with pCDNA3.1 or pCDNA3.1-Kaiso. The KBS-mutated constructs were negative controls. Renilla luciferase reporter pRL-TK was an internal control. The untreated cells served as a blank. Relative luciferase activity is presented as mean ± SD. *p

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

Anti-apoptotic effect of Kaiso in H 2 O 2 treated endothelial cells. Cells were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA and cell viability was analyzed at 0, 12, 24, 36, and 48 h after transfection using CCK8 method ( A ). Cells were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA and were cultured at 37 °C for 48 h. Then, cells were treated with 400 μM H 2 O 2 for 0, 2, 4, 8, and 12 h and cell viability was analyzed at the end of each time period using CCK8 method ( B ). HUVECs ( C ) and HMEC-1s ( D ) were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA and were cultured at 37 °C for 48 h. Cells were then treated with 400 μM H 2 O 2 for 8 h and cell apoptosis was examined using Annexin V/Propidium iodide (PI) flowcytometry analysis. The untreated cells served as a blank. Values are presented as mean ± SD, *p
Figure Legend Snippet: Anti-apoptotic effect of Kaiso in H 2 O 2 treated endothelial cells. Cells were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA and cell viability was analyzed at 0, 12, 24, 36, and 48 h after transfection using CCK8 method ( A ). Cells were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA and were cultured at 37 °C for 48 h. Then, cells were treated with 400 μM H 2 O 2 for 0, 2, 4, 8, and 12 h and cell viability was analyzed at the end of each time period using CCK8 method ( B ). HUVECs ( C ) and HMEC-1s ( D ) were transfected with pCDNA3.1, pCDNA3.1-Kaiso, NC-siRNA or Kaiso-siRNA and were cultured at 37 °C for 48 h. Cells were then treated with 400 μM H 2 O 2 for 8 h and cell apoptosis was examined using Annexin V/Propidium iodide (PI) flowcytometry analysis. The untreated cells served as a blank. Values are presented as mean ± SD, *p

Techniques Used: Transfection, Cell Culture

36) Product Images from "PTTG/securin activates expression of p53 and modulates its function"

Article Title: PTTG/securin activates expression of p53 and modulates its function

Journal: Molecular Cancer

doi: 10.1186/1476-4598-3-18

Electrophoretic mobility shift assays show the binding of c-myc protein to the c-myc/max sequence. A : Nuclear extract prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 gene promoter sequence carrying a normal c-myc/max binding site. Addition of a 20-fold molar excess of specific unlabeled DNA resulted in almost complete disappearance of the DNA-protein complex (lane 3). An arrow indicates the specific DNA-protein complex. B : Nuclear extracts prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 promoter sequence carrying a normal c-myc/max binding site. Addition of antibody directed against the N-terminal of c-myc resulted in a supershift (lane 3, indicated by an arrow) whereas no supershift was obtained when the [ 32 P]-labeled-D-172/-89 c-myc sequence was used as a probe (lane 5). Addition of antibodies directed against the C-terminal of c-myc did not result in supershift (lanes 4 6) when either of the probes was used in the binding reaction indicating that C-terminal of c-myc is not assessable. N.S indicates non-specific complex.
Figure Legend Snippet: Electrophoretic mobility shift assays show the binding of c-myc protein to the c-myc/max sequence. A : Nuclear extract prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 gene promoter sequence carrying a normal c-myc/max binding site. Addition of a 20-fold molar excess of specific unlabeled DNA resulted in almost complete disappearance of the DNA-protein complex (lane 3). An arrow indicates the specific DNA-protein complex. B : Nuclear extracts prepared from HEK293 cells transfected either with pcDNA3.1 (lane 1) or pcDNA3.1- PTTG (lane 2) and [ 32 P]-labeled p53 promoter sequence carrying a normal c-myc/max binding site. Addition of antibody directed against the N-terminal of c-myc resulted in a supershift (lane 3, indicated by an arrow) whereas no supershift was obtained when the [ 32 P]-labeled-D-172/-89 c-myc sequence was used as a probe (lane 5). Addition of antibodies directed against the C-terminal of c-myc did not result in supershift (lanes 4 6) when either of the probes was used in the binding reaction indicating that C-terminal of c-myc is not assessable. N.S indicates non-specific complex.

Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Transfection, Labeling

Overexpression of PTTG activates the expression of bax by upregulating p53 expression. A: MCF7 cells (open bars) and HEK293 cells (solid bars) were co-transfected with the bax promoter and increasing amounts of the pCDNA3.1- PTTG expression vector. Transfections were performed in duplicate and the results are expressed as the mean ± S.E.M of four independent experiments (*, p
Figure Legend Snippet: Overexpression of PTTG activates the expression of bax by upregulating p53 expression. A: MCF7 cells (open bars) and HEK293 cells (solid bars) were co-transfected with the bax promoter and increasing amounts of the pCDNA3.1- PTTG expression vector. Transfections were performed in duplicate and the results are expressed as the mean ± S.E.M of four independent experiments (*, p

Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation

Mapping of the PTTG interacting region in the p53 promoter sequence. HEK293 cells (open bars) and PC3 cells (solid bars) were co-transfected with various 5' and 3' deleted constructs of p53 promoter and pcDNA3.1- PTTG . The sequence of the p53 promoter construct (p53/-172/-89) containing the c-myc/max binding sequence is shown. The '*' indicates the position of c-myc/max binding sequence. Transfections were performed in duplicate and the results are expressed as mean ± S.E.M of four independent experiments (*, p
Figure Legend Snippet: Mapping of the PTTG interacting region in the p53 promoter sequence. HEK293 cells (open bars) and PC3 cells (solid bars) were co-transfected with various 5' and 3' deleted constructs of p53 promoter and pcDNA3.1- PTTG . The sequence of the p53 promoter construct (p53/-172/-89) containing the c-myc/max binding sequence is shown. The '*' indicates the position of c-myc/max binding sequence. Transfections were performed in duplicate and the results are expressed as mean ± S.E.M of four independent experiments (*, p

Techniques Used: Sequencing, Transfection, Construct, Binding Assay

Activation of the p53 promoter by PTTG is mediated through the c-myc/max sequence. HEK293 (open bars) and PC3 (solid bars) were co-transfected with the pcDNA3.1- PTTG and p53/-172/-89 (wild-type myc/max binding sequence) or the p53/-172/-89-Δ- c-myc (mutated myc/max binding sequence) promoter construct. Transfections were performed in duplicate and the results are expressed as mean ± S.E.M of four independent experiments (*, p
Figure Legend Snippet: Activation of the p53 promoter by PTTG is mediated through the c-myc/max sequence. HEK293 (open bars) and PC3 (solid bars) were co-transfected with the pcDNA3.1- PTTG and p53/-172/-89 (wild-type myc/max binding sequence) or the p53/-172/-89-Δ- c-myc (mutated myc/max binding sequence) promoter construct. Transfections were performed in duplicate and the results are expressed as mean ± S.E.M of four independent experiments (*, p

Techniques Used: Activation Assay, Sequencing, Transfection, Binding Assay, Construct

Expression of PTTG and p53 proteins in MCF7, HEK293 and PC3 cells transfected with either pcDNA 3.1 (Lane 1) or pcDNA3.1- PTTG cDNA (lane 2). A : Expression of PTTG and p53 proteins was analyzed by western blot analysis using a PTTG - or p53 -specific antibody as described in Methods. A β-Actin antibody was used as a control to determine the variation in protein concentration and loading. B : Double immunocytochemical analysis of HEK293 cells for the co-expression of PTTG and p53 proteins. HEK293 cells were transiently transfected with PTTG cDNA. After 48 hours of transfection, the cells were subjected to immunofluorescence analysis using PTTG -specific antiserum and a p53 monoclonal antibody. Panel A: Cells transfected with pcDNA3.1 vector. Panel B: Cells transfected with pcDNA3.1- PTTG cDNA. 1, pre-immune serum; 2, PTTG antiserum; 3, p53-specific monoclonal antibody, and 4, overlay of 2 and 3 showing co-expression of PTTG and p53 proteins.
Figure Legend Snippet: Expression of PTTG and p53 proteins in MCF7, HEK293 and PC3 cells transfected with either pcDNA 3.1 (Lane 1) or pcDNA3.1- PTTG cDNA (lane 2). A : Expression of PTTG and p53 proteins was analyzed by western blot analysis using a PTTG - or p53 -specific antibody as described in Methods. A β-Actin antibody was used as a control to determine the variation in protein concentration and loading. B : Double immunocytochemical analysis of HEK293 cells for the co-expression of PTTG and p53 proteins. HEK293 cells were transiently transfected with PTTG cDNA. After 48 hours of transfection, the cells were subjected to immunofluorescence analysis using PTTG -specific antiserum and a p53 monoclonal antibody. Panel A: Cells transfected with pcDNA3.1 vector. Panel B: Cells transfected with pcDNA3.1- PTTG cDNA. 1, pre-immune serum; 2, PTTG antiserum; 3, p53-specific monoclonal antibody, and 4, overlay of 2 and 3 showing co-expression of PTTG and p53 proteins.

Techniques Used: Expressing, Transfection, Western Blot, Protein Concentration, Immunofluorescence, Plasmid Preparation

37) Product Images from "Regulation of the Association of the PAF53/PAF49 Heterodimer with RNA Polymerase I"

Article Title: Regulation of the Association of the PAF53/PAF49 Heterodimer with RNA Polymerase I

Journal: Gene

doi: 10.1016/j.gene.2014.09.022

PAF49 is acetylated. 293T cells were transfected with pcDNA3.1 containing inserts that would drive the indicated proteins. Forty-eight hours post transfection, whole cell lysates were prepared and the FLAG-tagged proteins purified by immunoaffinity pull-down
Figure Legend Snippet: PAF49 is acetylated. 293T cells were transfected with pcDNA3.1 containing inserts that would drive the indicated proteins. Forty-eight hours post transfection, whole cell lysates were prepared and the FLAG-tagged proteins purified by immunoaffinity pull-down

Techniques Used: Transfection, Purification

38) Product Images from "FBXL19-AS1 exerts oncogenic function by sponging miR-431-5p to regulate RAF1 expression in lung cancer"

Article Title: FBXL19-AS1 exerts oncogenic function by sponging miR-431-5p to regulate RAF1 expression in lung cancer

Journal: Bioscience Reports

doi: 10.1042/BSR20181804

MiR-431-5p directly targets RAF1 and is negatively correlated to RAF1 expression ( A ) The predicted binding sites of miR-431-5p and RAF1 3′-UTR, and mutant sites in mutant-type RAF1 reporter. ( B ) RAF1 (Wt) or RAF1 (Mut) reporter were co-transfected with miR-NC, miR-431-5p mimic or miR-431-5p mimic +pcDNA3.1/FBXL19-AS1 into A549 and H1299 cells. After 48 h, relative luciferase activity was detected by using luciferase reporter assay. ( C ) RNA pull-down assay was performed to further confirm the binding ability between RAF1 and miR-431-5p in A549 and H1299 cells. ( D ) After 48 h of transfection, the protein expression of RAF1 was measured by western blot assay. ( E ) RT-qPCR results showed that overexpression of RAF1 reduced the expression of miR-431-5p. ( F ) RT-qPCR results showed that expression of RAF1 was higher in tumor tissues than in adjacent non-tumor tissues. ( G ) Spearman’s correlation analysis demonstrated the negative correlation between expression of RAF1 and miR-431-5p. ( H ) Spearman’s correlation analysis showed the positive correlation between expression of FBXL19-AS1 and RAF1. Error bars represent the mean ± SD of at least three independent experiments. * P
Figure Legend Snippet: MiR-431-5p directly targets RAF1 and is negatively correlated to RAF1 expression ( A ) The predicted binding sites of miR-431-5p and RAF1 3′-UTR, and mutant sites in mutant-type RAF1 reporter. ( B ) RAF1 (Wt) or RAF1 (Mut) reporter were co-transfected with miR-NC, miR-431-5p mimic or miR-431-5p mimic +pcDNA3.1/FBXL19-AS1 into A549 and H1299 cells. After 48 h, relative luciferase activity was detected by using luciferase reporter assay. ( C ) RNA pull-down assay was performed to further confirm the binding ability between RAF1 and miR-431-5p in A549 and H1299 cells. ( D ) After 48 h of transfection, the protein expression of RAF1 was measured by western blot assay. ( E ) RT-qPCR results showed that overexpression of RAF1 reduced the expression of miR-431-5p. ( F ) RT-qPCR results showed that expression of RAF1 was higher in tumor tissues than in adjacent non-tumor tissues. ( G ) Spearman’s correlation analysis demonstrated the negative correlation between expression of RAF1 and miR-431-5p. ( H ) Spearman’s correlation analysis showed the positive correlation between expression of FBXL19-AS1 and RAF1. Error bars represent the mean ± SD of at least three independent experiments. * P

Techniques Used: Expressing, Binding Assay, Mutagenesis, Transfection, Luciferase, Activity Assay, Reporter Assay, Pull Down Assay, Western Blot, Quantitative RT-PCR, Over Expression

39) Product Images from "The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain"

Article Title: The novel monoclonal antibody 9F5 reveals expression of a fragment of GPNMB/osteoactivin processed by furin‐like protease(s) in a subpopulation of microglia in neonatal rat brain

Journal: Glia

doi: 10.1002/glia.23034

Overexpression of furin cDNA increases the expression level of 9F5 antigen. COS‐7 cells were cotransfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 or pcDNA3.1‐ Gpnmb /pcDNA3.1‐furin vector. A : At 72 hr after transfection, cells were double‐stained with 9F5 (red) and anti‐GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means ± SEM ( n = 3). Fluorescence intensities (9F5 and anti‐GPNMB) in cells transfected with pcDNA3.1‐ Gpnmb /pcDNA3.1were set at 100%. * P
Figure Legend Snippet: Overexpression of furin cDNA increases the expression level of 9F5 antigen. COS‐7 cells were cotransfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 or pcDNA3.1‐ Gpnmb /pcDNA3.1‐furin vector. A : At 72 hr after transfection, cells were double‐stained with 9F5 (red) and anti‐GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means ± SEM ( n = 3). Fluorescence intensities (9F5 and anti‐GPNMB) in cells transfected with pcDNA3.1‐ Gpnmb /pcDNA3.1were set at 100%. * P

Techniques Used: Over Expression, Expressing, Plasmid Preparation, Transfection, Staining, Fluorescence

A furin siRNA inhibits the expression level of 9F5 antigen. HEK293 cells were cotransfected with pcDNA3.1‐ Gpnmb /control small interfering RNA (siRNA) or pcDNA3.1‐ Gpnmb /furin siRNA. A : At 72 hr after transfection, cells were double‐stained with 9F5 (red) and anti‐GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means ± SEM ( n = 3–4). Fluorescence intensities (9F5 and anti‐GPNMB) in cells transfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 were set at 100%. ** P
Figure Legend Snippet: A furin siRNA inhibits the expression level of 9F5 antigen. HEK293 cells were cotransfected with pcDNA3.1‐ Gpnmb /control small interfering RNA (siRNA) or pcDNA3.1‐ Gpnmb /furin siRNA. A : At 72 hr after transfection, cells were double‐stained with 9F5 (red) and anti‐GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means ± SEM ( n = 3–4). Fluorescence intensities (9F5 and anti‐GPNMB) in cells transfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 were set at 100%. ** P

Techniques Used: Expressing, Small Interfering RNA, Transfection, Staining, Fluorescence

Recognition by 9F5 of GPNMB protein in rat Gpnmb cDNA‐transfected COS‐7 cells. A–C : COS‐7 cells were transfected with pcDNA3.1‐ Gpnmb or a pcDNA3.1 empty vector for 72 hr. A: Cells were double‐stained with 9F5 and a commercially available goat anti‐mouse GPNMB polyclonal antibody. d, h : Phase‐contrast images. B: Cell lysates were from COS‐7 cells transfected with pcDNA3.1‐ Gpnmb (lane 1) or pcDNA3.1 empty vector (lane 2) or were from type 1 MG (lane 3). Rat GPNMB proteins were cross‐reacted with a goat anti‐mouse GPNMB antibody. Arrows indicate specific staining. C: Lysates of cells transfected with pcDNA3.1‐ Gpnmb or pcDNA3.1 empty vector were immunoprecipitated by using 9F5 (under DTT (−) condition) and then immunoblotted (IB) with the anti‐GPNMB antibody (under DTT (+) condition). Lysates from rat type 1 MG was used as a positive control (P.C.) for immunoblotting with anti‐GPNMB antibody. Lower panel shows the IgG (9F5 or control IgG) that was eluted from Protein G column by SDS‐PAGE sampling buffer without DTT. D : WB analysis of rat type 1 MG reactions with 9F5 and anti‐GPNMB antibody. E : Lysates of rat type 1 MG were immunoprecipitated via anti‐GPNMB antibody and then immunoblotted by using 9F5. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com .]
Figure Legend Snippet: Recognition by 9F5 of GPNMB protein in rat Gpnmb cDNA‐transfected COS‐7 cells. A–C : COS‐7 cells were transfected with pcDNA3.1‐ Gpnmb or a pcDNA3.1 empty vector for 72 hr. A: Cells were double‐stained with 9F5 and a commercially available goat anti‐mouse GPNMB polyclonal antibody. d, h : Phase‐contrast images. B: Cell lysates were from COS‐7 cells transfected with pcDNA3.1‐ Gpnmb (lane 1) or pcDNA3.1 empty vector (lane 2) or were from type 1 MG (lane 3). Rat GPNMB proteins were cross‐reacted with a goat anti‐mouse GPNMB antibody. Arrows indicate specific staining. C: Lysates of cells transfected with pcDNA3.1‐ Gpnmb or pcDNA3.1 empty vector were immunoprecipitated by using 9F5 (under DTT (−) condition) and then immunoblotted (IB) with the anti‐GPNMB antibody (under DTT (+) condition). Lysates from rat type 1 MG was used as a positive control (P.C.) for immunoblotting with anti‐GPNMB antibody. Lower panel shows the IgG (9F5 or control IgG) that was eluted from Protein G column by SDS‐PAGE sampling buffer without DTT. D : WB analysis of rat type 1 MG reactions with 9F5 and anti‐GPNMB antibody. E : Lysates of rat type 1 MG were immunoprecipitated via anti‐GPNMB antibody and then immunoblotted by using 9F5. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com .]

Techniques Used: Transfection, Plasmid Preparation, Staining, Immunoprecipitation, Positive Control, SDS Page, Sampling, Western Blot

A furin inhibitor decreases the expression level of 9F5 antigen. COS‐7 cells were cotransfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 or pcDNA3.1‐ Gpnmb /pcDNA3.1‐α1PDX vector. A : At 72 hr after transfection, cells were double‐stained with 9F5 (red) and anti‐GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means ± SEM ( n = 3–4). Fluorescence intensities (9F5 and anti‐GPNMB) in cells transfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 were set at 100%. ** P
Figure Legend Snippet: A furin inhibitor decreases the expression level of 9F5 antigen. COS‐7 cells were cotransfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 or pcDNA3.1‐ Gpnmb /pcDNA3.1‐α1PDX vector. A : At 72 hr after transfection, cells were double‐stained with 9F5 (red) and anti‐GPNMB (green) antibodies. B : The results shown in A were quantified and are given as means ± SEM ( n = 3–4). Fluorescence intensities (9F5 and anti‐GPNMB) in cells transfected with pcDNA3.1‐ Gpnmb /pcDNA3.1 were set at 100%. ** P

Techniques Used: Expressing, Plasmid Preparation, Transfection, Staining, Fluorescence

40) Product Images from "Rv2346c enhances mycobacterial survival within macrophages by inhibiting TNF-α and IL-6 production via the p38/miRNA/NF-κB pathway"

Article Title: Rv2346c enhances mycobacterial survival within macrophages by inhibiting TNF-α and IL-6 production via the p38/miRNA/NF-κB pathway

Journal: Emerging Microbes & Infections

doi: 10.1038/s41426-018-0162-6

Effect of p38 on the levels of miR-155 and miR-99b, p65 expression and cytokine production. U937 and RAW264.7 cells were transfected with a control vector (indicated with NC), pcDNA3.1/p38 cDNA (indicated with p38), a nonspecific siRNA (indicated with sh-NC) or a specific siRNA directed against p38 (indicated with sh-p38). After 24 h post transfection, total RNA was extracted. miR-155 and miR-99b were detected via qRT-PCR. The results are representative of three independent experiments. Data are presented as the means ± SD. * P
Figure Legend Snippet: Effect of p38 on the levels of miR-155 and miR-99b, p65 expression and cytokine production. U937 and RAW264.7 cells were transfected with a control vector (indicated with NC), pcDNA3.1/p38 cDNA (indicated with p38), a nonspecific siRNA (indicated with sh-NC) or a specific siRNA directed against p38 (indicated with sh-p38). After 24 h post transfection, total RNA was extracted. miR-155 and miR-99b were detected via qRT-PCR. The results are representative of three independent experiments. Data are presented as the means ± SD. * P

Techniques Used: Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR

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Transfection:

Article Title: CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts
Article Snippet: .. For transfection, human CCN1 cDNA expression vector was cloned into pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA), as described previously (Quan et al. ). .. Human skin dermal fibroblasts were transiently transfected by electroporation (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer’s protocol.

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Amplification:

Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo
Article Snippet: .. The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene. .. Transfection was performed using the Lipofectamine™ 2000 transfection reagent (invitrogen) according to the manufacturer's instructions.

Polymerase Chain Reaction:

Article Title: Identification and characterization of novel PAX8 mutations in Congenital Hypothyroidism(CH) in a Chinese population
Article Snippet: .. PCR products were cloned into the pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA) using KpnI and XbaI restriction sites introduced into the primers to obtain the wild-type (WT) PAX8-pCDNA3.1( PAX8WT-pCDNA3.1). .. Mutations were introduced using the Quick Change Mutagenesis kit (Transgene Biotech, Beijing, China), following the manufacturer's protocol, and the following primers: PAX8 D94N-pCDNA3.1 construct, 5′-GTGGAGAAGATTGGGAACTACAAACG-3′ and 5′-TCCCAATCTTCTCCACCACCTTGGGG-3′; and PAX8 G41V-pCDNA3.1 construct, 5′-ACCTGGCCCACCAGGTTGTAAGGCCC-3′ and 5′-ACCTGGTGGGCCAGGTCTACGATGCG-3′.

Article Title: A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus
Article Snippet: .. These PCR fragments were cloned into the pcDNA3.1 expression vector (Invitrogen) and designated as KSHV/gRta. .. Primer A and primer B were used to generate PCR products from total BC-1 DNA in a separate reaction.

Expressing:

Article Title: Identification and characterization of novel PAX8 mutations in Congenital Hypothyroidism(CH) in a Chinese population
Article Snippet: .. PCR products were cloned into the pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA) using KpnI and XbaI restriction sites introduced into the primers to obtain the wild-type (WT) PAX8-pCDNA3.1( PAX8WT-pCDNA3.1). .. Mutations were introduced using the Quick Change Mutagenesis kit (Transgene Biotech, Beijing, China), following the manufacturer's protocol, and the following primers: PAX8 D94N-pCDNA3.1 construct, 5′-GTGGAGAAGATTGGGAACTACAAACG-3′ and 5′-TCCCAATCTTCTCCACCACCTTGGGG-3′; and PAX8 G41V-pCDNA3.1 construct, 5′-ACCTGGCCCACCAGGTTGTAAGGCCC-3′ and 5′-ACCTGGTGGGCCAGGTCTACGATGCG-3′.

Article Title: Inhibition of mitochondrial respiration by nitric oxide rapidly stimulates cytoprotective GLUT3-mediated glucose uptake through 5?-AMP-activated protein kinase
Article Snippet: .. Full-length GLUT3 or GLUT1 cDNAs, digested from pBS-rG3, were also subcloned into the Eco RI site of the pcDNA3 mammalian expression vector (Invitrogen, Madrid, Spain; pcDNA3-G3). ..

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras
Article Snippet: .. Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously described . .. For construction of DsRed-H-Ras, full-length human H-Ras cDNA was amplified by PCR and cloned with DsRed-Monomer into the pcDNA3.1 vector.

Article Title: CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts
Article Snippet: .. For transfection, human CCN1 cDNA expression vector was cloned into pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA), as described previously (Quan et al. ). .. Human skin dermal fibroblasts were transiently transfected by electroporation (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer’s protocol.

Article Title: A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus
Article Snippet: .. These PCR fragments were cloned into the pcDNA3.1 expression vector (Invitrogen) and designated as KSHV/gRta. .. Primer A and primer B were used to generate PCR products from total BC-1 DNA in a separate reaction.

Article Title: GPC3 reduces cell proliferation in renal carcinoma cell lines
Article Snippet: .. Transfection The pcDNA3.1/GPC3 expression vector and pcDNA3.1 (empty vector) were transfected into ACHN and 786-O cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. .. RNA extraction and qRT-PCR Total RNA was extracted using TRIzol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions.

Sequencing:

Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo
Article Snippet: .. The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene. .. Transfection was performed using the Lipofectamine™ 2000 transfection reagent (invitrogen) according to the manufacturer's instructions.

Plasmid Preparation:

Article Title: Identification and characterization of novel PAX8 mutations in Congenital Hypothyroidism(CH) in a Chinese population
Article Snippet: .. PCR products were cloned into the pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA, USA) using KpnI and XbaI restriction sites introduced into the primers to obtain the wild-type (WT) PAX8-pCDNA3.1( PAX8WT-pCDNA3.1). .. Mutations were introduced using the Quick Change Mutagenesis kit (Transgene Biotech, Beijing, China), following the manufacturer's protocol, and the following primers: PAX8 D94N-pCDNA3.1 construct, 5′-GTGGAGAAGATTGGGAACTACAAACG-3′ and 5′-TCCCAATCTTCTCCACCACCTTGGGG-3′; and PAX8 G41V-pCDNA3.1 construct, 5′-ACCTGGCCCACCAGGTTGTAAGGCCC-3′ and 5′-ACCTGGTGGGCCAGGTCTACGATGCG-3′.

Article Title: MicroRNA-34a Inhibits the Proliferation and Metastasis of Osteosarcoma Cells Both In Vitro and In Vivo
Article Snippet: .. The correct sequences of amplified fragment were verified by sequencing, double digested with HindIII and BamHI, and cloned into pcDNA3.1 vector (Invitrogen), carrying neomycin resistance gene. .. Transfection was performed using the Lipofectamine™ 2000 transfection reagent (invitrogen) according to the manufacturer's instructions.

Article Title: Inhibition of mitochondrial respiration by nitric oxide rapidly stimulates cytoprotective GLUT3-mediated glucose uptake through 5?-AMP-activated protein kinase
Article Snippet: .. Full-length GLUT3 or GLUT1 cDNAs, digested from pBS-rG3, were also subcloned into the Eco RI site of the pcDNA3 mammalian expression vector (Invitrogen, Madrid, Spain; pcDNA3-G3). ..

Article Title: UBIAD1 suppresses the proliferation of bladder carcinoma cells by regulating H-Ras intracellular trafficking via interaction with the C-terminal domain of H-Ras
Article Snippet: .. Enhanced green fluorescent protein (EGFP) vectors pEGFP-N1, pEGFP-C1, pCasper3-BG (TAGBFP-GFP), mammalian expression vector pcDNA3.1 and TAGBFP were obtained from Invitrogen. pDsRed-Golgi vector was obtained from Clontech, previously described . .. For construction of DsRed-H-Ras, full-length human H-Ras cDNA was amplified by PCR and cloned with DsRed-Monomer into the pcDNA3.1 vector.

Article Title: Transient mismatch repair gene transfection for functional analysis of genetic hMLH1 and hMSH2 variants
Article Snippet: .. The complete wild type cDNA for hMSH2 was subcloned from laboratory isolates into the pcDNA3.1+ vector (Invitrogen, Groningen, Netherlands), placing the cDNA under the control of the CMV promoter. .. Inserts for hMSH2 wt cDNA were amplified using primers bearing BamHI (sense) or XhoI (antisense) restriction sites and ligated in the appropriate sites of the vector.

Article Title: CCN1 contributes to skin connective tissue aging by inducing age-associated secretory phenotype in human skin dermal fibroblasts
Article Snippet: .. For transfection, human CCN1 cDNA expression vector was cloned into pCDNA3.1 expression vector (Invitrogen, Carlsbad, CA), as described previously (Quan et al. ). .. Human skin dermal fibroblasts were transiently transfected by electroporation (Amaxa Biosystems, Gaithersburg, MD) according to the manufacturer’s protocol.

Article Title: A viral gene that activates lytic cycle expression of Kaposi's sarcoma-associated herpesvirus
Article Snippet: .. These PCR fragments were cloned into the pcDNA3.1 expression vector (Invitrogen) and designated as KSHV/gRta. .. Primer A and primer B were used to generate PCR products from total BC-1 DNA in a separate reaction.

Article Title: GPC3 reduces cell proliferation in renal carcinoma cell lines
Article Snippet: .. Transfection The pcDNA3.1/GPC3 expression vector and pcDNA3.1 (empty vector) were transfected into ACHN and 786-O cell lines using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. .. RNA extraction and qRT-PCR Total RNA was extracted using TRIzol reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer’s instructions.

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  • 93
    Thermo Fisher pcdna3 1 linc00702
    Overexpression of <t>LINC00702</t> inhibited NSCLC cell proliferation and invasion via inducing apoptosis in vitro . ( A ) Quantitative RT-PCR analysis of relative LINC00702 expression levels in NCI-H441 and PC-9 transfected with <t>pcDNA3.1,</t> pcDNA3.1-LINC00702 or NC. ( B, C ) CCK-8 assays were used to evaluate the effect of LINC00702 on proliferation ability of NCI-H441 and PC-9 cells. NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to CCK-8 assays ( D ) EdU staining was performed in NCI-H441 and PC-9 cells. ( E, F ) Colony formation assays were used to evaluate the effect of LINC00702 on anchor-independent growth ability of NCI-H441 and PC-9 cells. NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to colony formation assays. ( G, H ). NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to transwell assay. ( I, J ) Flow cytometry was used to evaluate the effect of LINC00702 on apoptosis of NCI-H441 and PC-9 cells. **P
    Pcdna3 1 Linc00702, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 linc00702/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 linc00702 - by Bioz Stars, 2020-09
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    92
    Thermo Fisher pcdna3 1
    ATF4 induced CSE transcription via direct binding to a cis regulatory intronic site. A and B , luciferase activity resulting from HEK cells transfected with constructs as indicated together with (empty vector control plasmid) <t>pCDNA3.1</t> or overexpressed ATF4. RLU , relative light units. n = 4. *, p
    Pcdna3 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1996 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1/product/Thermo Fisher
    Average 92 stars, based on 1996 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 - by Bioz Stars, 2020-09
    92/100 stars
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    92
    Thermo Fisher pcdna3 1 rip1
    Knockdown of <t>RIP1</t> in GBC cells impaired TNF-α-mediated tube formation in HDLECs. Notes: Red fluorescent probe-labeled HDLECs were cocultured with the established NOZ or GBC-SD cell groups (control, LV-siNC, and LV-siRIP1) and analyzed using a fluorescence microscope, and the number of tubes was calculated. ( A , C ) 50 ng/mL of recombinant human TNF-α enhanced tube formation in HDLECs, and siRIP1 impaired this enhancement by TNF-α. Transfection of <t>PcDNA3.1-RIP1</t> vector into LV-siRIP1 cells reversed this impairment. ( B , D ) Supplementation of recombinant human VEGF-C protein (50 ng/mL) into the LV-siRIP1 cell group reversed the impairment of TNF-α-enhanced tube formation. C and D; n=3, mean±SEM * p
    Pcdna3 1 Rip1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcdna3 1 rip1/product/Thermo Fisher
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    pcdna3 1 rip1 - by Bioz Stars, 2020-09
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    84
    Thermo Fisher pcdna3 1 ogr1
    <t>OGR1</t> inhibits A549 cell migration via CK2 ( A ) OGR1 inhibits A549 cell migration but presence of CK2 inhibitor, CX4945 abrogated the effect of OGR1 but not the presence of NEP inhibitor, thiophan (THP). A549 cell were transfected with pDNA3.1-OGR1 or <t>pcDNA3.1</t> (Vector) in presence of CK2 and NEP inhibitors. Cell migration was analysed and image was taken hourly. ( B ) To measure the area of wound, the length of scratches were measured on the light microscope and plotted on graph. Wound area of control 0 hr was taken as 100% and means SD. P-values were calculated comparing with the control values of the same hrs of treatment by students’ “t” test. The bar (Red) indicates the scale of 100µm. Bars indicate SD, * indicates p-value
    Pcdna3 1 Ogr1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Overexpression of LINC00702 inhibited NSCLC cell proliferation and invasion via inducing apoptosis in vitro . ( A ) Quantitative RT-PCR analysis of relative LINC00702 expression levels in NCI-H441 and PC-9 transfected with pcDNA3.1, pcDNA3.1-LINC00702 or NC. ( B, C ) CCK-8 assays were used to evaluate the effect of LINC00702 on proliferation ability of NCI-H441 and PC-9 cells. NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to CCK-8 assays ( D ) EdU staining was performed in NCI-H441 and PC-9 cells. ( E, F ) Colony formation assays were used to evaluate the effect of LINC00702 on anchor-independent growth ability of NCI-H441 and PC-9 cells. NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to colony formation assays. ( G, H ). NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to transwell assay. ( I, J ) Flow cytometry was used to evaluate the effect of LINC00702 on apoptosis of NCI-H441 and PC-9 cells. **P

    Journal: Aging (Albany NY)

    Article Title: LINC00702 suppresses proliferation and invasion in non-small cell lung cancer through regulating miR-510/PTEN axis

    doi: 10.18632/aging.101846

    Figure Lengend Snippet: Overexpression of LINC00702 inhibited NSCLC cell proliferation and invasion via inducing apoptosis in vitro . ( A ) Quantitative RT-PCR analysis of relative LINC00702 expression levels in NCI-H441 and PC-9 transfected with pcDNA3.1, pcDNA3.1-LINC00702 or NC. ( B, C ) CCK-8 assays were used to evaluate the effect of LINC00702 on proliferation ability of NCI-H441 and PC-9 cells. NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to CCK-8 assays ( D ) EdU staining was performed in NCI-H441 and PC-9 cells. ( E, F ) Colony formation assays were used to evaluate the effect of LINC00702 on anchor-independent growth ability of NCI-H441 and PC-9 cells. NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to colony formation assays. ( G, H ). NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h and subjected to transwell assay. ( I, J ) Flow cytometry was used to evaluate the effect of LINC00702 on apoptosis of NCI-H441 and PC-9 cells. **P

    Article Snippet: EdU staining NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h, then cells were subjected to EdU Alexa Fluor™ 555 staining (Thermo Fisher Scientific, USA) according to the protocol of manufacture.

    Techniques: Over Expression, In Vitro, Quantitative RT-PCR, Expressing, Transfection, CCK-8 Assay, Staining, Transwell Assay, Flow Cytometry, Cytometry

    MiR-510 directly targeted PTEN gene. ( A ) The predicted binding sites in PTEN and miR-510 and the mutant sequence of PTEN. ( B ) A firefly luciferase reporter containing either wild-type (WT) or mutant (MT) PTEN was transfected with miR-510 mimics (20 nM) in NCI-H441 cells. ( C ) A firefly luciferase reporter containing either wild-type (WT) or mutant (MT) PTEN was transfected miR-510 mimics (20 nM) in PC-9 cells. ( D ) Quantitative RT-PCR analysis of relative PTEN expression levels in NSCLC cells transfected with control mimics or miR-510 mimics. ( E ) Quantitative RT-PCR analysis of relative PTEN expression levels in NSCLC cells transfected with pcDNA3.1-LINC00702 or/and miR-510 mimics. *P

    Journal: Aging (Albany NY)

    Article Title: LINC00702 suppresses proliferation and invasion in non-small cell lung cancer through regulating miR-510/PTEN axis

    doi: 10.18632/aging.101846

    Figure Lengend Snippet: MiR-510 directly targeted PTEN gene. ( A ) The predicted binding sites in PTEN and miR-510 and the mutant sequence of PTEN. ( B ) A firefly luciferase reporter containing either wild-type (WT) or mutant (MT) PTEN was transfected with miR-510 mimics (20 nM) in NCI-H441 cells. ( C ) A firefly luciferase reporter containing either wild-type (WT) or mutant (MT) PTEN was transfected miR-510 mimics (20 nM) in PC-9 cells. ( D ) Quantitative RT-PCR analysis of relative PTEN expression levels in NSCLC cells transfected with control mimics or miR-510 mimics. ( E ) Quantitative RT-PCR analysis of relative PTEN expression levels in NSCLC cells transfected with pcDNA3.1-LINC00702 or/and miR-510 mimics. *P

    Article Snippet: EdU staining NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h, then cells were subjected to EdU Alexa Fluor™ 555 staining (Thermo Fisher Scientific, USA) according to the protocol of manufacture.

    Techniques: Binding Assay, Mutagenesis, Sequencing, Luciferase, Transfection, Quantitative RT-PCR, Expressing

    LINC00702 functioned as a ceRNA for miR-510 in NSCLC cells. ( A ) Quantitative RT-PCR analysis of relative miRNAs expression levels in NCI-H441 cells transfected with pcDNA3.1 or pcDNA3.1-LINC00702. ( B ) Quantitative RT-PCR analysis of relative miRNAs expression levels in PC-9 cells transfected with pcDNA3.1 or pcDNA3.1-LINC00702. ( C ) Quantitative RT-PCR analysis of relative miRNA-510 expression levels in 40 pairs NSCLC tumor and adjacent tissues. P

    Journal: Aging (Albany NY)

    Article Title: LINC00702 suppresses proliferation and invasion in non-small cell lung cancer through regulating miR-510/PTEN axis

    doi: 10.18632/aging.101846

    Figure Lengend Snippet: LINC00702 functioned as a ceRNA for miR-510 in NSCLC cells. ( A ) Quantitative RT-PCR analysis of relative miRNAs expression levels in NCI-H441 cells transfected with pcDNA3.1 or pcDNA3.1-LINC00702. ( B ) Quantitative RT-PCR analysis of relative miRNAs expression levels in PC-9 cells transfected with pcDNA3.1 or pcDNA3.1-LINC00702. ( C ) Quantitative RT-PCR analysis of relative miRNA-510 expression levels in 40 pairs NSCLC tumor and adjacent tissues. P

    Article Snippet: EdU staining NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h, then cells were subjected to EdU Alexa Fluor™ 555 staining (Thermo Fisher Scientific, USA) according to the protocol of manufacture.

    Techniques: Quantitative RT-PCR, Expressing, Transfection

    PTEN was a target gene of miR-510. ( A ) Western blot analysis of the expressions of PTEN, p-Akt and Akt in NSCLC cells co-transfected with pcDNA3.1-LINC00702 or/and miR-510 mimics. ( B ) Relative PTEN protein levels in NCI-H441 and PC-9 cells. ( C ) Relative p-Akt protein levels in NCI-H441 and PC-9 cells. ( D ) The expression of PTEN in NSCLC cells co-transfected with pcDNA3.1-LINC00702 or/and miR-510 mimics. ( E ) Relative PTEN expression were calculated. *P

    Journal: Aging (Albany NY)

    Article Title: LINC00702 suppresses proliferation and invasion in non-small cell lung cancer through regulating miR-510/PTEN axis

    doi: 10.18632/aging.101846

    Figure Lengend Snippet: PTEN was a target gene of miR-510. ( A ) Western blot analysis of the expressions of PTEN, p-Akt and Akt in NSCLC cells co-transfected with pcDNA3.1-LINC00702 or/and miR-510 mimics. ( B ) Relative PTEN protein levels in NCI-H441 and PC-9 cells. ( C ) Relative p-Akt protein levels in NCI-H441 and PC-9 cells. ( D ) The expression of PTEN in NSCLC cells co-transfected with pcDNA3.1-LINC00702 or/and miR-510 mimics. ( E ) Relative PTEN expression were calculated. *P

    Article Snippet: EdU staining NCI-H441 and PC-9 cells were transfected with pcDNA3.1 or pcDNA3.1-LINC00702 for 72 h, then cells were subjected to EdU Alexa Fluor™ 555 staining (Thermo Fisher Scientific, USA) according to the protocol of manufacture.

    Techniques: Western Blot, Transfection, Expressing

    ATF4 induced CSE transcription via direct binding to a cis regulatory intronic site. A and B , luciferase activity resulting from HEK cells transfected with constructs as indicated together with (empty vector control plasmid) pCDNA3.1 or overexpressed ATF4. RLU , relative light units. n = 4. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling *

    doi: 10.1074/jbc.M115.685578

    Figure Lengend Snippet: ATF4 induced CSE transcription via direct binding to a cis regulatory intronic site. A and B , luciferase activity resulting from HEK cells transfected with constructs as indicated together with (empty vector control plasmid) pCDNA3.1 or overexpressed ATF4. RLU , relative light units. n = 4. *, p

    Article Snippet: For luciferase assays, HEK cells were transfected in 24-well plates with a constant amount of 900 ng of total DNA composed of 300 ng of test plasmid, 300 ng of reference plasmid (pRL-TK, Promega), and 300 ng of pCDNA3.1 or pATF4 using the TurboFect transfection reagent (2 μl/well; Thermo Scientific).

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

    Nox4 regulated CSE expression via ATF4. A , representative Western blot and quantitative densitometric analyses of ATF4 protein expression and the corresponding QPCR analyses of CSE mRNA expression in HUVECs after 24-h ATF4 or control pCDNA3.1 overexpression. A.U. , absorbance units. B , representative Western blot and quantitative densitometric analyses of ATF4 protein expression in HUVEC after 48 h of treatment with ATF4-targeted siRNA (siATF4) or control siRNA (siScram) together with 24 h Nox4 or β-gal ( B Gal ) overexpression as indicated. C , QPCR analyses of ATF4 and CSE mRNA expression in HUVECs after treatments as in B. D , representative Western blot and corresponding densitometric analyses of ATF4 protein expression in CMECs isolated from WT and eNox4 Tg mice. All data are normalized to β-actin mRNA and protein expression. n = 3; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling *

    doi: 10.1074/jbc.M115.685578

    Figure Lengend Snippet: Nox4 regulated CSE expression via ATF4. A , representative Western blot and quantitative densitometric analyses of ATF4 protein expression and the corresponding QPCR analyses of CSE mRNA expression in HUVECs after 24-h ATF4 or control pCDNA3.1 overexpression. A.U. , absorbance units. B , representative Western blot and quantitative densitometric analyses of ATF4 protein expression in HUVEC after 48 h of treatment with ATF4-targeted siRNA (siATF4) or control siRNA (siScram) together with 24 h Nox4 or β-gal ( B Gal ) overexpression as indicated. C , QPCR analyses of ATF4 and CSE mRNA expression in HUVECs after treatments as in B. D , representative Western blot and corresponding densitometric analyses of ATF4 protein expression in CMECs isolated from WT and eNox4 Tg mice. All data are normalized to β-actin mRNA and protein expression. n = 3; *, p

    Article Snippet: For luciferase assays, HEK cells were transfected in 24-well plates with a constant amount of 900 ng of total DNA composed of 300 ng of test plasmid, 300 ng of reference plasmid (pRL-TK, Promega), and 300 ng of pCDNA3.1 or pATF4 using the TurboFect transfection reagent (2 μl/well; Thermo Scientific).

    Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Over Expression, Isolation, Mouse Assay

    Knockdown of RIP1 in GBC cells impaired TNF-α-mediated tube formation in HDLECs. Notes: Red fluorescent probe-labeled HDLECs were cocultured with the established NOZ or GBC-SD cell groups (control, LV-siNC, and LV-siRIP1) and analyzed using a fluorescence microscope, and the number of tubes was calculated. ( A , C ) 50 ng/mL of recombinant human TNF-α enhanced tube formation in HDLECs, and siRIP1 impaired this enhancement by TNF-α. Transfection of PcDNA3.1-RIP1 vector into LV-siRIP1 cells reversed this impairment. ( B , D ) Supplementation of recombinant human VEGF-C protein (50 ng/mL) into the LV-siRIP1 cell group reversed the impairment of TNF-α-enhanced tube formation. C and D; n=3, mean±SEM * p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: Knockdown of RIP1 in GBC cells impaired TNF-α-mediated tube formation in HDLECs. Notes: Red fluorescent probe-labeled HDLECs were cocultured with the established NOZ or GBC-SD cell groups (control, LV-siNC, and LV-siRIP1) and analyzed using a fluorescence microscope, and the number of tubes was calculated. ( A , C ) 50 ng/mL of recombinant human TNF-α enhanced tube formation in HDLECs, and siRIP1 impaired this enhancement by TNF-α. Transfection of PcDNA3.1-RIP1 vector into LV-siRIP1 cells reversed this impairment. ( B , D ) Supplementation of recombinant human VEGF-C protein (50 ng/mL) into the LV-siRIP1 cell group reversed the impairment of TNF-α-enhanced tube formation. C and D; n=3, mean±SEM * p

    Article Snippet: Transfection of the siIκBα, PcDNA3.1-RIP1, pRL-TK, pGL3-Basic, pGL3B-332, and NF-κB-luc reporter plasmids was conducted in Opti-MEM medium (Thermo Fisher Scientific) using Lipofectamine reagent (Thermo Fisher Scientific).

    Techniques: Labeling, Fluorescence, Microscopy, Recombinant, Transfection, Plasmid Preparation

    RIP1 is essential for TNF-α-mediated NF-κB activation. Notes: ( A ) NF-κB-luciferase activity was examined in the siNC and siRIP1 cell groups after being stimulated with 50 ng/ml of recombinant human TNF-α or left unstimulated for 24 h. Transfection of PcDNA3.1-RIP1 vector into the siRIP1 cell groups reversed the impairment of TNF-α-mediated NF-κB activation. ( B ) Western blot analyses of RIP1, iκBα, and p-iκBα expression in protein extracts from the siNC and siRIP cell groups that were stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. ( C–E ) Transfection of silκBα into NOZ or GBC-SD cells effectively inhibited iκBα mRNA and protein expression. (F) NF-κB-luciferase activity assays showed that knockdown of lκBα could reverse the impairment of TNF-α-mediated NF-κB activation in the siRIP1 cell groups. (G) immunoprecipitation analysis showed that TAK1 and NEMO are associated with RIP1. B, C, D and F; n=3, mean±SEM; * p

    Journal: OncoTargets and therapy

    Article Title: RIP1 regulates TNF-α-mediated lymphangiogenesis and lymphatic metastasis in gallbladder cancer by modulating the NF-κB-VEGF-C pathway

    doi: 10.2147/OTT.S159026

    Figure Lengend Snippet: RIP1 is essential for TNF-α-mediated NF-κB activation. Notes: ( A ) NF-κB-luciferase activity was examined in the siNC and siRIP1 cell groups after being stimulated with 50 ng/ml of recombinant human TNF-α or left unstimulated for 24 h. Transfection of PcDNA3.1-RIP1 vector into the siRIP1 cell groups reversed the impairment of TNF-α-mediated NF-κB activation. ( B ) Western blot analyses of RIP1, iκBα, and p-iκBα expression in protein extracts from the siNC and siRIP cell groups that were stimulated with 50 ng/mL of recombinant human TNF-α or left unstimulated for 24 h. ( C–E ) Transfection of silκBα into NOZ or GBC-SD cells effectively inhibited iκBα mRNA and protein expression. (F) NF-κB-luciferase activity assays showed that knockdown of lκBα could reverse the impairment of TNF-α-mediated NF-κB activation in the siRIP1 cell groups. (G) immunoprecipitation analysis showed that TAK1 and NEMO are associated with RIP1. B, C, D and F; n=3, mean±SEM; * p

    Article Snippet: Transfection of the siIκBα, PcDNA3.1-RIP1, pRL-TK, pGL3-Basic, pGL3B-332, and NF-κB-luc reporter plasmids was conducted in Opti-MEM medium (Thermo Fisher Scientific) using Lipofectamine reagent (Thermo Fisher Scientific).

    Techniques: Activation Assay, Luciferase, Activity Assay, Recombinant, Transfection, Plasmid Preparation, Western Blot, Expressing, Immunoprecipitation

    OGR1 inhibits A549 cell migration via CK2 ( A ) OGR1 inhibits A549 cell migration but presence of CK2 inhibitor, CX4945 abrogated the effect of OGR1 but not the presence of NEP inhibitor, thiophan (THP). A549 cell were transfected with pDNA3.1-OGR1 or pcDNA3.1 (Vector) in presence of CK2 and NEP inhibitors. Cell migration was analysed and image was taken hourly. ( B ) To measure the area of wound, the length of scratches were measured on the light microscope and plotted on graph. Wound area of control 0 hr was taken as 100% and means SD. P-values were calculated comparing with the control values of the same hrs of treatment by students’ “t” test. The bar (Red) indicates the scale of 100µm. Bars indicate SD, * indicates p-value

    Journal: bioRxiv

    Article Title: Ovarian cancer G protein-coupled receptor 1 inhibits A549 cells Migration through Casein kinase 2α intronless gene

    doi: 10.1101/556720

    Figure Lengend Snippet: OGR1 inhibits A549 cell migration via CK2 ( A ) OGR1 inhibits A549 cell migration but presence of CK2 inhibitor, CX4945 abrogated the effect of OGR1 but not the presence of NEP inhibitor, thiophan (THP). A549 cell were transfected with pDNA3.1-OGR1 or pcDNA3.1 (Vector) in presence of CK2 and NEP inhibitors. Cell migration was analysed and image was taken hourly. ( B ) To measure the area of wound, the length of scratches were measured on the light microscope and plotted on graph. Wound area of control 0 hr was taken as 100% and means SD. P-values were calculated comparing with the control values of the same hrs of treatment by students’ “t” test. The bar (Red) indicates the scale of 100µm. Bars indicate SD, * indicates p-value

    Article Snippet: A549 cells were transiently transfected with 1.0–1.5μg plasmids; pcDNA3.1-OGR1 and empty vector (pcDNA3.1) or co-transfected empty vector or pcDNA3.1-OGR1 with pcDNA3.1-cdcT17N and pcDNA3.1-RacT17N using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol.

    Techniques: Migration, Transfection, Plasmid Preparation, Light Microscopy

    Role of Rac/CDC42 and MAPK pathways in regulation of CK2α genes induced by OGR1 (A) A549 cells were co-transfected with OGR1 or pcDNA3.1 (Vector) and negative dominant mutants of Rac (pcDNA3.1-RacT17N) or negative dominant mutants of CDC42 (pcDNA3.1-cdcT17N). Semiquantitative RT-PCR of was performed to analyse CSNK2A1 and CSNK2A3 transcript expression. β-actin was used as control for equal loading; Inhibition of Rac activity abrogated up-regulation of CSNK2A3 induced by OGR1 (B) A549 cells were also transfected with pDNA3.1-OGR1 or pcDNA3.1 (Vector) in presence of MAPK kinase inhibitors. Semi-quantitative RT-PCR of was performed to analyse CSNK2A1 and CSNK2A3 transcript expression. β-actin was used as control for equal loading; inhibition of JNK and p38 using specific inhibitors, SP600125 and SB2203580 respectively, abrogated the up-regulate of CSNK2A3 induced by OGR1 but not by inhibition of ERK (FR180204) whereas inhibition of ERK, JNK and p38 does not significantly affect the expression of CSNK2A1 in A549.

    Journal: bioRxiv

    Article Title: Ovarian cancer G protein-coupled receptor 1 inhibits A549 cells Migration through Casein kinase 2α intronless gene

    doi: 10.1101/556720

    Figure Lengend Snippet: Role of Rac/CDC42 and MAPK pathways in regulation of CK2α genes induced by OGR1 (A) A549 cells were co-transfected with OGR1 or pcDNA3.1 (Vector) and negative dominant mutants of Rac (pcDNA3.1-RacT17N) or negative dominant mutants of CDC42 (pcDNA3.1-cdcT17N). Semiquantitative RT-PCR of was performed to analyse CSNK2A1 and CSNK2A3 transcript expression. β-actin was used as control for equal loading; Inhibition of Rac activity abrogated up-regulation of CSNK2A3 induced by OGR1 (B) A549 cells were also transfected with pDNA3.1-OGR1 or pcDNA3.1 (Vector) in presence of MAPK kinase inhibitors. Semi-quantitative RT-PCR of was performed to analyse CSNK2A1 and CSNK2A3 transcript expression. β-actin was used as control for equal loading; inhibition of JNK and p38 using specific inhibitors, SP600125 and SB2203580 respectively, abrogated the up-regulate of CSNK2A3 induced by OGR1 but not by inhibition of ERK (FR180204) whereas inhibition of ERK, JNK and p38 does not significantly affect the expression of CSNK2A1 in A549.

    Article Snippet: A549 cells were transiently transfected with 1.0–1.5μg plasmids; pcDNA3.1-OGR1 and empty vector (pcDNA3.1) or co-transfected empty vector or pcDNA3.1-OGR1 with pcDNA3.1-cdcT17N and pcDNA3.1-RacT17N using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol.

    Techniques: Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Expressing, Inhibition, Activity Assay, Quantitative RT-PCR

    OGR1 up-regulates CSNK2A3 (CK2α intronless gene) and NEP but not CSNK2A1 in A549 ( A ) A549 cells were transfected with OGR1 (OGR1) or pcDNA3.1 (Vector), transcript expressions of CSNK2A1, CSNK2A3 and NEP were analysed in the by semi-quantitative RT-PCR. β-actin was used as control for equal loading. ( B ) RNA bands intensities for CSNK2A1 and CSNK2A3 were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented in graph. ( C) Protein expressions of NEP and CK2α were assayed by Western blotting with specific antibodies and β-actin was used as control for equal landing, ( D ) protein band intensities were analysed by using ‒a Image Acquisition and Analysis software (BioRad, USA) and presented as graph. Experiments were repeated thrice. Statistical significance between control (Vector) and OGR1 was calculated using student’s “t” test. Bars indicates SD, ** indicates p-value

    Journal: bioRxiv

    Article Title: Ovarian cancer G protein-coupled receptor 1 inhibits A549 cells Migration through Casein kinase 2α intronless gene

    doi: 10.1101/556720

    Figure Lengend Snippet: OGR1 up-regulates CSNK2A3 (CK2α intronless gene) and NEP but not CSNK2A1 in A549 ( A ) A549 cells were transfected with OGR1 (OGR1) or pcDNA3.1 (Vector), transcript expressions of CSNK2A1, CSNK2A3 and NEP were analysed in the by semi-quantitative RT-PCR. β-actin was used as control for equal loading. ( B ) RNA bands intensities for CSNK2A1 and CSNK2A3 were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented in graph. ( C) Protein expressions of NEP and CK2α were assayed by Western blotting with specific antibodies and β-actin was used as control for equal landing, ( D ) protein band intensities were analysed by using ‒a Image Acquisition and Analysis software (BioRad, USA) and presented as graph. Experiments were repeated thrice. Statistical significance between control (Vector) and OGR1 was calculated using student’s “t” test. Bars indicates SD, ** indicates p-value

    Article Snippet: A549 cells were transiently transfected with 1.0–1.5μg plasmids; pcDNA3.1-OGR1 and empty vector (pcDNA3.1) or co-transfected empty vector or pcDNA3.1-OGR1 with pcDNA3.1-cdcT17N and pcDNA3.1-RacT17N using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol.

    Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Software, Western Blot

    CK2 is upstream of NEP in the OGR1 signalling pathway Western blot assay of NEP and CK2α expression in presence or absence of specific inhibitors. ( A ) A549 cells were transfected with pDNA3.1-OGR1 (OGR1) or pcDNA3.1 (Vector) and NEP expression was analysed with anti NEP in presence or absence of CX-4945, CK2 inhibitor (CX4945) and (B) protein band intensities were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented as graph, ( C ) expression of CK2α was analysed with anti CK2 in presence or absence of NEP inhibitor, thiophan (THP) and ( D ) protein band intensities were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented as graph, ( E ) Semi Quantitaive PCR showing the expression of NEP and CK2 in presence or absence of Gi inhibitor PTX and ( F ) intensities of protein bands were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented in graph. Stastatical significance between treated and untreated was calculated using student’s “t” test. Bars indicate SD, * indicates p-value

    Journal: bioRxiv

    Article Title: Ovarian cancer G protein-coupled receptor 1 inhibits A549 cells Migration through Casein kinase 2α intronless gene

    doi: 10.1101/556720

    Figure Lengend Snippet: CK2 is upstream of NEP in the OGR1 signalling pathway Western blot assay of NEP and CK2α expression in presence or absence of specific inhibitors. ( A ) A549 cells were transfected with pDNA3.1-OGR1 (OGR1) or pcDNA3.1 (Vector) and NEP expression was analysed with anti NEP in presence or absence of CX-4945, CK2 inhibitor (CX4945) and (B) protein band intensities were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented as graph, ( C ) expression of CK2α was analysed with anti CK2 in presence or absence of NEP inhibitor, thiophan (THP) and ( D ) protein band intensities were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented as graph, ( E ) Semi Quantitaive PCR showing the expression of NEP and CK2 in presence or absence of Gi inhibitor PTX and ( F ) intensities of protein bands were analysed by using Image Acquisition and Analysis software (BioRad, USA) and presented in graph. Stastatical significance between treated and untreated was calculated using student’s “t” test. Bars indicate SD, * indicates p-value

    Article Snippet: A549 cells were transiently transfected with 1.0–1.5μg plasmids; pcDNA3.1-OGR1 and empty vector (pcDNA3.1) or co-transfected empty vector or pcDNA3.1-OGR1 with pcDNA3.1-cdcT17N and pcDNA3.1-RacT17N using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s protocol.

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