rat ddr2 cd167b gene orf cdna  (Sino Biological)


Bioz Verified Symbol Sino Biological is a verified supplier
Bioz Manufacturer Symbol Sino Biological manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Sino Biological rat ddr2 cd167b gene orf cdna
    <t>DDR2</t> regulates Ang II-mediated expression of AT1R in cardiac fibroblasts ( A ) RNAi-mediated silencing of DDR2 confirmed its role in regulating AT1R expression in Ang II-stimulated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with DDR2 siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. Validation of DDR2 silencing is also shown. *p
    Rat Ddr2 Cd167b Gene Orf Cdna, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat ddr2 cd167b gene orf cdna/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rat ddr2 cd167b gene orf cdna - by Bioz Stars, 2021-07
    86/100 stars

    Images

    1) Product Images from "Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling"

    Article Title: Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling

    Journal: bioRxiv

    doi: 10.1101/2021.05.06.442987

    DDR2 regulates Ang II-mediated expression of AT1R in cardiac fibroblasts ( A ) RNAi-mediated silencing of DDR2 confirmed its role in regulating AT1R expression in Ang II-stimulated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with DDR2 siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. Validation of DDR2 silencing is also shown. *p
    Figure Legend Snippet: DDR2 regulates Ang II-mediated expression of AT1R in cardiac fibroblasts ( A ) RNAi-mediated silencing of DDR2 confirmed its role in regulating AT1R expression in Ang II-stimulated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with DDR2 siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. Validation of DDR2 silencing is also shown. *p

    Techniques Used: Expressing, Transfection

    DDR2 mediates Ang II-dependent fibronectin expression in cardiac fibroblasts. (A) Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Angiotensin II (Ang II) (1μM). Protein was isolated at 12 h of Ang II treatment and subjected to western blot analysis for detection of fibronectin and DDR2, with β-actin as loading control. *p
    Figure Legend Snippet: DDR2 mediates Ang II-dependent fibronectin expression in cardiac fibroblasts. (A) Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Angiotensin II (Ang II) (1μM). Protein was isolated at 12 h of Ang II treatment and subjected to western blot analysis for detection of fibronectin and DDR2, with β-actin as loading control. *p

    Techniques Used: Expressing, Isolation, Western Blot

    Transcriptional regulation of fibronectin by DDR2-activated YAP transcription coactivator ( A ) Cardiac fibroblasts were treated with Verteporfin (10 μM) for 1 h prior to Ang II treatment. Cells were collected at 12 h post-Ang II treatment and fibronectin protein levels were examined, with β-actin as loading control. **p
    Figure Legend Snippet: Transcriptional regulation of fibronectin by DDR2-activated YAP transcription coactivator ( A ) Cardiac fibroblasts were treated with Verteporfin (10 μM) for 1 h prior to Ang II treatment. Cells were collected at 12 h post-Ang II treatment and fibronectin protein levels were examined, with β-actin as loading control. **p

    Techniques Used:

    2) Product Images from "Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts"

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    Journal: bioRxiv

    doi: 10.1101/857037

    Enhanced expression of DDR2 correlates with enhanced levels of SRF, cIAP2 and PCNA in freshly isolated cardiac fibroblasts from Spontaneously Hypertensive Rats. ( A ) Cardiac fibroblasts freshly isolated from 6-month old male SHR and Wistar rats were analysed by western blotting for levels of cIAP2, SRF, PCNA and DDR2 with β-actin as loading control. Significance was determined by Student’s t test, ** p
    Figure Legend Snippet: Enhanced expression of DDR2 correlates with enhanced levels of SRF, cIAP2 and PCNA in freshly isolated cardiac fibroblasts from Spontaneously Hypertensive Rats. ( A ) Cardiac fibroblasts freshly isolated from 6-month old male SHR and Wistar rats were analysed by western blotting for levels of cIAP2, SRF, PCNA and DDR2 with β-actin as loading control. Significance was determined by Student’s t test, ** p

    Techniques Used: Expressing, Isolation, Western Blot

    Regulation of p27: Post-translational regulation via SRF-dependent Skp2. ( A-G ) Sub-confluent cultures of cardiac fibroblasts were transfected with SRF siRNA ( A, F and G ), ERK1/2 siRNA ( C ) or DDR2 siRNA ( B, D and E ) (with Control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serum-deprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) SRF siRNA transfected cells were collected at 8h and analysed by western blotting for expression levels of Skp2, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p
    Figure Legend Snippet: Regulation of p27: Post-translational regulation via SRF-dependent Skp2. ( A-G ) Sub-confluent cultures of cardiac fibroblasts were transfected with SRF siRNA ( A, F and G ), ERK1/2 siRNA ( C ) or DDR2 siRNA ( B, D and E ) (with Control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serum-deprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) SRF siRNA transfected cells were collected at 8h and analysed by western blotting for expression levels of Skp2, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Techniques Used: Transfection, Western Blot, Expressing

    Regulation of p27 by DDR2: ii) Transcriptional regulation through modulating FoxO3a activity. ( A-E ) Sub-confluent cultures of cardiac fibroblasts were transfected with DDR2 siRNA ( A, B and C ) or ERK1/2 siRNA ( D and E ) (with control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serumdeprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) DDR2 siRNA-transfected cells were collected at 8h and analysed by western blotting for expression levels of Phospho-FoxO3a (T32)/Total FoxO3a, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p
    Figure Legend Snippet: Regulation of p27 by DDR2: ii) Transcriptional regulation through modulating FoxO3a activity. ( A-E ) Sub-confluent cultures of cardiac fibroblasts were transfected with DDR2 siRNA ( A, B and C ) or ERK1/2 siRNA ( D and E ) (with control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serumdeprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) DDR2 siRNA-transfected cells were collected at 8h and analysed by western blotting for expression levels of Phospho-FoxO3a (T32)/Total FoxO3a, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

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

    DDR2 over-expression facilitates G1-S transition in mitogen-deprived cardiac fibroblasts. ( A-E ) Cardiac fibroblasts transfected with DDR2 cDNA over-expression plasmid (DDR2 OE) or empty vector control (Control OE) were subjected to western blot analysis (with β-actin as loading control) or flow cytometric analysis. Validation of DDR2 overexpression is also shown. ( A ) Flow cytometric profile of G1-S transition, showing distribution of cells in each phase. Significance was determined by Student’s t test, **p
    Figure Legend Snippet: DDR2 over-expression facilitates G1-S transition in mitogen-deprived cardiac fibroblasts. ( A-E ) Cardiac fibroblasts transfected with DDR2 cDNA over-expression plasmid (DDR2 OE) or empty vector control (Control OE) were subjected to western blot analysis (with β-actin as loading control) or flow cytometric analysis. Validation of DDR2 overexpression is also shown. ( A ) Flow cytometric profile of G1-S transition, showing distribution of cells in each phase. Significance was determined by Student’s t test, **p

    Techniques Used: Over Expression, Transfection, Plasmid Preparation, Western Blot

    DDR2 mediates Ang II-stimulated expression of anti-apoptotic cIAP2 in cardiac fibroblasts. Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Ang II (1μM). (A) cIAP2 mRNA levels were determined by Taqman Real-time PCR analysis at 6h of Ang II treatment. β-actin served as the endogenous control. Significance was determined by Student’s t test, *p
    Figure Legend Snippet: DDR2 mediates Ang II-stimulated expression of anti-apoptotic cIAP2 in cardiac fibroblasts. Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Ang II (1μM). (A) cIAP2 mRNA levels were determined by Taqman Real-time PCR analysis at 6h of Ang II treatment. β-actin served as the endogenous control. Significance was determined by Student’s t test, *p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    DDR2 knockdown in mitogen-stimulated cardiac fibroblasts inhibits G1-S transition. ( A) Sub-confluent cultures of cardiac fibroblasts were serum deprived for 24h prior to treatment with Control (fresh M199 medium treatment), 25μM H 2 O 2 , Ang II (1μM) and 10% Fetal Calf Serum. Post treatment, cells were collected at 8h and analysed by western blotting for expression levels of PCNA and cyclin D1, with β-actin as loading control. Significance was determined by Student’s t test, (each treatment vs Control) **p
    Figure Legend Snippet: DDR2 knockdown in mitogen-stimulated cardiac fibroblasts inhibits G1-S transition. ( A) Sub-confluent cultures of cardiac fibroblasts were serum deprived for 24h prior to treatment with Control (fresh M199 medium treatment), 25μM H 2 O 2 , Ang II (1μM) and 10% Fetal Calf Serum. Post treatment, cells were collected at 8h and analysed by western blotting for expression levels of PCNA and cyclin D1, with β-actin as loading control. Significance was determined by Student’s t test, (each treatment vs Control) **p

    Techniques Used: Western Blot, Expressing

    DDR2-dependent cIAP2 expression protects cardiac fibroblasts against oxidative damage. ( A-B ) Effect of AT1 receptor antagonist, Candesartan, on H 2 O 2 -treated cardiac fibroblasts was analysed. Four sets of sub-confluent cultures of cardiac fibroblasts were serum-deprived for 24h: i) no treatment (control), ii) 25μM H 2 O 2 iii) pre-incubated with 10μM candesartan (AT1 receptor antagonist) for 1 h and was treated with 25μM H 2 O 2 iv) 10μM candesartan alone. ( A ) Cells were collected 12h post-H 2 O 2 addition and analysed by western blot for the expression of cIAP2, DDR2 and SRF, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, *p
    Figure Legend Snippet: DDR2-dependent cIAP2 expression protects cardiac fibroblasts against oxidative damage. ( A-B ) Effect of AT1 receptor antagonist, Candesartan, on H 2 O 2 -treated cardiac fibroblasts was analysed. Four sets of sub-confluent cultures of cardiac fibroblasts were serum-deprived for 24h: i) no treatment (control), ii) 25μM H 2 O 2 iii) pre-incubated with 10μM candesartan (AT1 receptor antagonist) for 1 h and was treated with 25μM H 2 O 2 iv) 10μM candesartan alone. ( A ) Cells were collected 12h post-H 2 O 2 addition and analysed by western blot for the expression of cIAP2, DDR2 and SRF, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, *p

    Techniques Used: Expressing, Incubation, Western Blot

    A schematic representation of the plausible molecular events that integrate apoptosis resistance and proliferation under the regulatory control of DDR2 in cardiac fibroblasts. In response to the stimulus (Ang II or 10% FCS), collagen type I-dependent activation of DDR2 leads to the activation of ERK1/2 MAPK that in turn activates SRF to: i) transcriptionally increase cIAP2, conferring apoptosis resistance, ii) increase Skp2 and promote Skp2-mediated post-translational degradation of p27, and iii) inactivate FoxO3a, through phosphorylation, to transcriptionally repress p27. Transcriptional and posttranslational inhibition of p27 results in Cyclin D1/CDK4/6 complex-dependent phosphorylation of Rb protein, facilitating the transcription of S-phase genes and G1-S transition.
    Figure Legend Snippet: A schematic representation of the plausible molecular events that integrate apoptosis resistance and proliferation under the regulatory control of DDR2 in cardiac fibroblasts. In response to the stimulus (Ang II or 10% FCS), collagen type I-dependent activation of DDR2 leads to the activation of ERK1/2 MAPK that in turn activates SRF to: i) transcriptionally increase cIAP2, conferring apoptosis resistance, ii) increase Skp2 and promote Skp2-mediated post-translational degradation of p27, and iii) inactivate FoxO3a, through phosphorylation, to transcriptionally repress p27. Transcriptional and posttranslational inhibition of p27 results in Cyclin D1/CDK4/6 complex-dependent phosphorylation of Rb protein, facilitating the transcription of S-phase genes and G1-S transition.

    Techniques Used: Activation Assay, Inhibition

    Obligate role of collagen type I in the activation (phosphorylation) of DDR2. ( A ). Validation blot for immunoprecipitation of DDR2. DDR2 was pulled down from cardiac fibroblast cell lysates using specific DDR2 anti-rabbit IgG (2ug antibody for 100ug of total cell lysate) and protein A magnetic beads. Western blot was performed to confirm the pull down of DDR2. In the blot, “input” indicates the fraction of total cell lysate before immunoprecipitation, and “unbound” the supernatant of the immunoprecipitated sample. Lane 3 is the immunoprecipitated DDR2 (DDR2 IP), showing DDR2 pull down. (B-D) Following serum deprivation for 24h, cardiac fibroblasts were divided into 4 groups and treated as follows: 1) Control + WRG-28 (2μM), 2) Control + DMSO, 3) Stimulus + WRG-28 (2μM), and 4) Stimulus + DMSO. Ang II (1μM) or H 2 O 2 (25μM) or 10% Fetal Calf Serum (10 %FCS) was used as the stimulus. Before treatment with the stimulus, the cells were pre-incubated with either WRG-28 (2μM) or vehicle DMSO for 45 min. Following preincubation, the cells were treated with the stimulus for 3, 6 and 12h and total DDR2 was immunoprecipitated (see also supplementary figure S2A) and subjected to western blot analysis of Phospho-tyrosine levels (activated DDR2) normalized to total DDR2 levels. ( B ) Phospho-tyrosine levels in immunoprecipitated DDR2 at 3, 6 and 12h of Ang II (1μM) treatment. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p
    Figure Legend Snippet: Obligate role of collagen type I in the activation (phosphorylation) of DDR2. ( A ). Validation blot for immunoprecipitation of DDR2. DDR2 was pulled down from cardiac fibroblast cell lysates using specific DDR2 anti-rabbit IgG (2ug antibody for 100ug of total cell lysate) and protein A magnetic beads. Western blot was performed to confirm the pull down of DDR2. In the blot, “input” indicates the fraction of total cell lysate before immunoprecipitation, and “unbound” the supernatant of the immunoprecipitated sample. Lane 3 is the immunoprecipitated DDR2 (DDR2 IP), showing DDR2 pull down. (B-D) Following serum deprivation for 24h, cardiac fibroblasts were divided into 4 groups and treated as follows: 1) Control + WRG-28 (2μM), 2) Control + DMSO, 3) Stimulus + WRG-28 (2μM), and 4) Stimulus + DMSO. Ang II (1μM) or H 2 O 2 (25μM) or 10% Fetal Calf Serum (10 %FCS) was used as the stimulus. Before treatment with the stimulus, the cells were pre-incubated with either WRG-28 (2μM) or vehicle DMSO for 45 min. Following preincubation, the cells were treated with the stimulus for 3, 6 and 12h and total DDR2 was immunoprecipitated (see also supplementary figure S2A) and subjected to western blot analysis of Phospho-tyrosine levels (activated DDR2) normalized to total DDR2 levels. ( B ) Phospho-tyrosine levels in immunoprecipitated DDR2 at 3, 6 and 12h of Ang II (1μM) treatment. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Techniques Used: Activation Assay, Immunoprecipitation, Magnetic Beads, Western Blot, Incubation

    DDR2-dependent ERK1/2 MAPK activation acts via SRF to transcriptionally upregulate cIAP2 expression in Ang II-stimulated cardiac fibroblasts. ( A ) Sub-confluent quiescent cultures of cardiac fibroblasts were pre-treated with CCG-1423(10μM) for 45 min prior to treatment with Ang II (1μM) for 12h. cIAP2, SRF and DDR2 protein expression was examined by western blot analysis, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p
    Figure Legend Snippet: DDR2-dependent ERK1/2 MAPK activation acts via SRF to transcriptionally upregulate cIAP2 expression in Ang II-stimulated cardiac fibroblasts. ( A ) Sub-confluent quiescent cultures of cardiac fibroblasts were pre-treated with CCG-1423(10μM) for 45 min prior to treatment with Ang II (1μM) for 12h. cIAP2, SRF and DDR2 protein expression was examined by western blot analysis, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Techniques Used: Activation Assay, Expressing, Western Blot

    Related Articles

    Expressing:

    Article Title: Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II–stimulated cardiac fibroblasts
    Article Snippet: Integrin-β1 and α-SMA siRNAs were custom-designed from Eurogentec (Liege, Belgium). .. The rat DDR2/CD167b gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China). .. The TRPC6 ( ) rat tagged ORF clone was from Origene (Rockville, MD).

    Article Title: Discoidin domain Receptor 2: A determinant of metabolic syndrome-associated arterial fibrosis in non-human primates
    Article Snippet: Opti-MEM and fetal bovine serum were from GIBCO (Waltham, USA). .. Rat DDR2/CD167b Gene ORF cDNA clone expression plasmid and FLAG-tagged Rat DDR2 cDNA clone expression plasmid were obtained from Sino Biologicals (Beijing, China). .. All cell culture ware was purchased from BD Falcon (Corning, USA).

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts
    Article Snippet: Signal Silence ® P44/42 MAPK (ERK1/2) siRNA was obtained from Cell Signaling Technology (Danvers, MA, USA). .. The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA. .. Opti-MEM and fetal Calf serum (FCS) were from GIBCO (Waltham, MA, USA).

    Plasmid Preparation:

    Article Title: Collagen receptor cross-talk determines α-smooth muscle actin-dependent collagen gene expression in angiotensin II–stimulated cardiac fibroblasts
    Article Snippet: Integrin-β1 and α-SMA siRNAs were custom-designed from Eurogentec (Liege, Belgium). .. The rat DDR2/CD167b gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China). .. The TRPC6 ( ) rat tagged ORF clone was from Origene (Rockville, MD).

    Article Title: Discoidin domain Receptor 2: A determinant of metabolic syndrome-associated arterial fibrosis in non-human primates
    Article Snippet: Opti-MEM and fetal bovine serum were from GIBCO (Waltham, USA). .. Rat DDR2/CD167b Gene ORF cDNA clone expression plasmid and FLAG-tagged Rat DDR2 cDNA clone expression plasmid were obtained from Sino Biologicals (Beijing, China). .. All cell culture ware was purchased from BD Falcon (Corning, USA).

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts
    Article Snippet: Signal Silence ® P44/42 MAPK (ERK1/2) siRNA was obtained from Cell Signaling Technology (Danvers, MA, USA). .. The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA. .. Opti-MEM and fetal Calf serum (FCS) were from GIBCO (Waltham, MA, USA).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    Sino Biological rat ddr2 cd167b gene orf cdna
    <t>DDR2</t> regulates Ang II-mediated expression of AT1R in cardiac fibroblasts ( A ) RNAi-mediated silencing of DDR2 confirmed its role in regulating AT1R expression in Ang II-stimulated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with DDR2 siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. Validation of DDR2 silencing is also shown. *p
    Rat Ddr2 Cd167b Gene Orf Cdna, supplied by Sino Biological, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rat ddr2 cd167b gene orf cdna/product/Sino Biological
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rat ddr2 cd167b gene orf cdna - by Bioz Stars, 2021-07
    86/100 stars
      Buy from Supplier

    Image Search Results


    DDR2 regulates Ang II-mediated expression of AT1R in cardiac fibroblasts ( A ) RNAi-mediated silencing of DDR2 confirmed its role in regulating AT1R expression in Ang II-stimulated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with DDR2 siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. Validation of DDR2 silencing is also shown. *p

    Journal: bioRxiv

    Article Title: Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling

    doi: 10.1101/2021.05.06.442987

    Figure Lengend Snippet: DDR2 regulates Ang II-mediated expression of AT1R in cardiac fibroblasts ( A ) RNAi-mediated silencing of DDR2 confirmed its role in regulating AT1R expression in Ang II-stimulated cardiac fibroblasts. Cardiac fibroblasts were transiently transfected with DDR2 siRNA (5 pmol) or control (scrambled) siRNA prior to treatment with Ang II for 12 h. AT1R protein expression was examined, with β-actin as loading control. Validation of DDR2 silencing is also shown. *p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China).

    Techniques: Expressing, Transfection

    DDR2 mediates Ang II-dependent fibronectin expression in cardiac fibroblasts. (A) Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Angiotensin II (Ang II) (1μM). Protein was isolated at 12 h of Ang II treatment and subjected to western blot analysis for detection of fibronectin and DDR2, with β-actin as loading control. *p

    Journal: bioRxiv

    Article Title: Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling

    doi: 10.1101/2021.05.06.442987

    Figure Lengend Snippet: DDR2 mediates Ang II-dependent fibronectin expression in cardiac fibroblasts. (A) Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Angiotensin II (Ang II) (1μM). Protein was isolated at 12 h of Ang II treatment and subjected to western blot analysis for detection of fibronectin and DDR2, with β-actin as loading control. *p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China).

    Techniques: Expressing, Isolation, Western Blot

    Transcriptional regulation of fibronectin by DDR2-activated YAP transcription coactivator ( A ) Cardiac fibroblasts were treated with Verteporfin (10 μM) for 1 h prior to Ang II treatment. Cells were collected at 12 h post-Ang II treatment and fibronectin protein levels were examined, with β-actin as loading control. **p

    Journal: bioRxiv

    Article Title: Discoidin Domain Receptor 2 regulates AT1 receptor expression in Angiotensin II-stimulated cardiac fibroblasts via fibronectin-dependent Integrin-β1 signalling

    doi: 10.1101/2021.05.06.442987

    Figure Lengend Snippet: Transcriptional regulation of fibronectin by DDR2-activated YAP transcription coactivator ( A ) Cardiac fibroblasts were treated with Verteporfin (10 μM) for 1 h prior to Ang II treatment. Cells were collected at 12 h post-Ang II treatment and fibronectin protein levels were examined, with β-actin as loading control. **p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China).

    Techniques:

    Enhanced expression of DDR2 correlates with enhanced levels of SRF, cIAP2 and PCNA in freshly isolated cardiac fibroblasts from Spontaneously Hypertensive Rats. ( A ) Cardiac fibroblasts freshly isolated from 6-month old male SHR and Wistar rats were analysed by western blotting for levels of cIAP2, SRF, PCNA and DDR2 with β-actin as loading control. Significance was determined by Student’s t test, ** p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: Enhanced expression of DDR2 correlates with enhanced levels of SRF, cIAP2 and PCNA in freshly isolated cardiac fibroblasts from Spontaneously Hypertensive Rats. ( A ) Cardiac fibroblasts freshly isolated from 6-month old male SHR and Wistar rats were analysed by western blotting for levels of cIAP2, SRF, PCNA and DDR2 with β-actin as loading control. Significance was determined by Student’s t test, ** p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Expressing, Isolation, Western Blot

    Regulation of p27: Post-translational regulation via SRF-dependent Skp2. ( A-G ) Sub-confluent cultures of cardiac fibroblasts were transfected with SRF siRNA ( A, F and G ), ERK1/2 siRNA ( C ) or DDR2 siRNA ( B, D and E ) (with Control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serum-deprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) SRF siRNA transfected cells were collected at 8h and analysed by western blotting for expression levels of Skp2, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: Regulation of p27: Post-translational regulation via SRF-dependent Skp2. ( A-G ) Sub-confluent cultures of cardiac fibroblasts were transfected with SRF siRNA ( A, F and G ), ERK1/2 siRNA ( C ) or DDR2 siRNA ( B, D and E ) (with Control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serum-deprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) SRF siRNA transfected cells were collected at 8h and analysed by western blotting for expression levels of Skp2, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Transfection, Western Blot, Expressing

    Regulation of p27 by DDR2: ii) Transcriptional regulation through modulating FoxO3a activity. ( A-E ) Sub-confluent cultures of cardiac fibroblasts were transfected with DDR2 siRNA ( A, B and C ) or ERK1/2 siRNA ( D and E ) (with control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serumdeprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) DDR2 siRNA-transfected cells were collected at 8h and analysed by western blotting for expression levels of Phospho-FoxO3a (T32)/Total FoxO3a, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: Regulation of p27 by DDR2: ii) Transcriptional regulation through modulating FoxO3a activity. ( A-E ) Sub-confluent cultures of cardiac fibroblasts were transfected with DDR2 siRNA ( A, B and C ) or ERK1/2 siRNA ( D and E ) (with control siRNA) and, following revival for 12h in 10% serum-supplemented medium, the cells were serumdeprived for synchronization. Post-synchronization, the cells were exposed to 10% Fetal Calf Serum. ( A ) DDR2 siRNA-transfected cells were collected at 8h and analysed by western blotting for expression levels of Phospho-FoxO3a (T32)/Total FoxO3a, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Activity Assay, Transfection, Western Blot, Expressing

    DDR2 over-expression facilitates G1-S transition in mitogen-deprived cardiac fibroblasts. ( A-E ) Cardiac fibroblasts transfected with DDR2 cDNA over-expression plasmid (DDR2 OE) or empty vector control (Control OE) were subjected to western blot analysis (with β-actin as loading control) or flow cytometric analysis. Validation of DDR2 overexpression is also shown. ( A ) Flow cytometric profile of G1-S transition, showing distribution of cells in each phase. Significance was determined by Student’s t test, **p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: DDR2 over-expression facilitates G1-S transition in mitogen-deprived cardiac fibroblasts. ( A-E ) Cardiac fibroblasts transfected with DDR2 cDNA over-expression plasmid (DDR2 OE) or empty vector control (Control OE) were subjected to western blot analysis (with β-actin as loading control) or flow cytometric analysis. Validation of DDR2 overexpression is also shown. ( A ) Flow cytometric profile of G1-S transition, showing distribution of cells in each phase. Significance was determined by Student’s t test, **p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot

    DDR2 mediates Ang II-stimulated expression of anti-apoptotic cIAP2 in cardiac fibroblasts. Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Ang II (1μM). (A) cIAP2 mRNA levels were determined by Taqman Real-time PCR analysis at 6h of Ang II treatment. β-actin served as the endogenous control. Significance was determined by Student’s t test, *p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: DDR2 mediates Ang II-stimulated expression of anti-apoptotic cIAP2 in cardiac fibroblasts. Sub-confluent quiescent cultures of cardiac fibroblasts were stimulated with Ang II (1μM). (A) cIAP2 mRNA levels were determined by Taqman Real-time PCR analysis at 6h of Ang II treatment. β-actin served as the endogenous control. Significance was determined by Student’s t test, *p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    DDR2 knockdown in mitogen-stimulated cardiac fibroblasts inhibits G1-S transition. ( A) Sub-confluent cultures of cardiac fibroblasts were serum deprived for 24h prior to treatment with Control (fresh M199 medium treatment), 25μM H 2 O 2 , Ang II (1μM) and 10% Fetal Calf Serum. Post treatment, cells were collected at 8h and analysed by western blotting for expression levels of PCNA and cyclin D1, with β-actin as loading control. Significance was determined by Student’s t test, (each treatment vs Control) **p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: DDR2 knockdown in mitogen-stimulated cardiac fibroblasts inhibits G1-S transition. ( A) Sub-confluent cultures of cardiac fibroblasts were serum deprived for 24h prior to treatment with Control (fresh M199 medium treatment), 25μM H 2 O 2 , Ang II (1μM) and 10% Fetal Calf Serum. Post treatment, cells were collected at 8h and analysed by western blotting for expression levels of PCNA and cyclin D1, with β-actin as loading control. Significance was determined by Student’s t test, (each treatment vs Control) **p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Western Blot, Expressing

    DDR2-dependent cIAP2 expression protects cardiac fibroblasts against oxidative damage. ( A-B ) Effect of AT1 receptor antagonist, Candesartan, on H 2 O 2 -treated cardiac fibroblasts was analysed. Four sets of sub-confluent cultures of cardiac fibroblasts were serum-deprived for 24h: i) no treatment (control), ii) 25μM H 2 O 2 iii) pre-incubated with 10μM candesartan (AT1 receptor antagonist) for 1 h and was treated with 25μM H 2 O 2 iv) 10μM candesartan alone. ( A ) Cells were collected 12h post-H 2 O 2 addition and analysed by western blot for the expression of cIAP2, DDR2 and SRF, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, *p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: DDR2-dependent cIAP2 expression protects cardiac fibroblasts against oxidative damage. ( A-B ) Effect of AT1 receptor antagonist, Candesartan, on H 2 O 2 -treated cardiac fibroblasts was analysed. Four sets of sub-confluent cultures of cardiac fibroblasts were serum-deprived for 24h: i) no treatment (control), ii) 25μM H 2 O 2 iii) pre-incubated with 10μM candesartan (AT1 receptor antagonist) for 1 h and was treated with 25μM H 2 O 2 iv) 10μM candesartan alone. ( A ) Cells were collected 12h post-H 2 O 2 addition and analysed by western blot for the expression of cIAP2, DDR2 and SRF, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, *p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Expressing, Incubation, Western Blot

    A schematic representation of the plausible molecular events that integrate apoptosis resistance and proliferation under the regulatory control of DDR2 in cardiac fibroblasts. In response to the stimulus (Ang II or 10% FCS), collagen type I-dependent activation of DDR2 leads to the activation of ERK1/2 MAPK that in turn activates SRF to: i) transcriptionally increase cIAP2, conferring apoptosis resistance, ii) increase Skp2 and promote Skp2-mediated post-translational degradation of p27, and iii) inactivate FoxO3a, through phosphorylation, to transcriptionally repress p27. Transcriptional and posttranslational inhibition of p27 results in Cyclin D1/CDK4/6 complex-dependent phosphorylation of Rb protein, facilitating the transcription of S-phase genes and G1-S transition.

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: A schematic representation of the plausible molecular events that integrate apoptosis resistance and proliferation under the regulatory control of DDR2 in cardiac fibroblasts. In response to the stimulus (Ang II or 10% FCS), collagen type I-dependent activation of DDR2 leads to the activation of ERK1/2 MAPK that in turn activates SRF to: i) transcriptionally increase cIAP2, conferring apoptosis resistance, ii) increase Skp2 and promote Skp2-mediated post-translational degradation of p27, and iii) inactivate FoxO3a, through phosphorylation, to transcriptionally repress p27. Transcriptional and posttranslational inhibition of p27 results in Cyclin D1/CDK4/6 complex-dependent phosphorylation of Rb protein, facilitating the transcription of S-phase genes and G1-S transition.

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Activation Assay, Inhibition

    Obligate role of collagen type I in the activation (phosphorylation) of DDR2. ( A ). Validation blot for immunoprecipitation of DDR2. DDR2 was pulled down from cardiac fibroblast cell lysates using specific DDR2 anti-rabbit IgG (2ug antibody for 100ug of total cell lysate) and protein A magnetic beads. Western blot was performed to confirm the pull down of DDR2. In the blot, “input” indicates the fraction of total cell lysate before immunoprecipitation, and “unbound” the supernatant of the immunoprecipitated sample. Lane 3 is the immunoprecipitated DDR2 (DDR2 IP), showing DDR2 pull down. (B-D) Following serum deprivation for 24h, cardiac fibroblasts were divided into 4 groups and treated as follows: 1) Control + WRG-28 (2μM), 2) Control + DMSO, 3) Stimulus + WRG-28 (2μM), and 4) Stimulus + DMSO. Ang II (1μM) or H 2 O 2 (25μM) or 10% Fetal Calf Serum (10 %FCS) was used as the stimulus. Before treatment with the stimulus, the cells were pre-incubated with either WRG-28 (2μM) or vehicle DMSO for 45 min. Following preincubation, the cells were treated with the stimulus for 3, 6 and 12h and total DDR2 was immunoprecipitated (see also supplementary figure S2A) and subjected to western blot analysis of Phospho-tyrosine levels (activated DDR2) normalized to total DDR2 levels. ( B ) Phospho-tyrosine levels in immunoprecipitated DDR2 at 3, 6 and 12h of Ang II (1μM) treatment. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: Obligate role of collagen type I in the activation (phosphorylation) of DDR2. ( A ). Validation blot for immunoprecipitation of DDR2. DDR2 was pulled down from cardiac fibroblast cell lysates using specific DDR2 anti-rabbit IgG (2ug antibody for 100ug of total cell lysate) and protein A magnetic beads. Western blot was performed to confirm the pull down of DDR2. In the blot, “input” indicates the fraction of total cell lysate before immunoprecipitation, and “unbound” the supernatant of the immunoprecipitated sample. Lane 3 is the immunoprecipitated DDR2 (DDR2 IP), showing DDR2 pull down. (B-D) Following serum deprivation for 24h, cardiac fibroblasts were divided into 4 groups and treated as follows: 1) Control + WRG-28 (2μM), 2) Control + DMSO, 3) Stimulus + WRG-28 (2μM), and 4) Stimulus + DMSO. Ang II (1μM) or H 2 O 2 (25μM) or 10% Fetal Calf Serum (10 %FCS) was used as the stimulus. Before treatment with the stimulus, the cells were pre-incubated with either WRG-28 (2μM) or vehicle DMSO for 45 min. Following preincubation, the cells were treated with the stimulus for 3, 6 and 12h and total DDR2 was immunoprecipitated (see also supplementary figure S2A) and subjected to western blot analysis of Phospho-tyrosine levels (activated DDR2) normalized to total DDR2 levels. ( B ) Phospho-tyrosine levels in immunoprecipitated DDR2 at 3, 6 and 12h of Ang II (1μM) treatment. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Activation Assay, Immunoprecipitation, Magnetic Beads, Western Blot, Incubation

    DDR2-dependent ERK1/2 MAPK activation acts via SRF to transcriptionally upregulate cIAP2 expression in Ang II-stimulated cardiac fibroblasts. ( A ) Sub-confluent quiescent cultures of cardiac fibroblasts were pre-treated with CCG-1423(10μM) for 45 min prior to treatment with Ang II (1μM) for 12h. cIAP2, SRF and DDR2 protein expression was examined by western blot analysis, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Journal: bioRxiv

    Article Title: Co-ordinated regulation of cell survival and cell cycle pathways by DDR2-dependent SRF transcription factor in cardiac fibroblasts

    doi: 10.1101/857037

    Figure Lengend Snippet: DDR2-dependent ERK1/2 MAPK activation acts via SRF to transcriptionally upregulate cIAP2 expression in Ang II-stimulated cardiac fibroblasts. ( A ) Sub-confluent quiescent cultures of cardiac fibroblasts were pre-treated with CCG-1423(10μM) for 45 min prior to treatment with Ang II (1μM) for 12h. cIAP2, SRF and DDR2 protein expression was examined by western blot analysis, with β-actin as loading control. Significance was determined by two-way ANOVA (Tukey’s multiple comparisons test, **p

    Article Snippet: The rat DDR2/CD167b Gene ORF cDNA clone expression plasmid was obtained from Sino Biologicals (Beijing, China) and Native ORF cIAP-2 clone in pCMV vector was purchased from Origene, Rockville, USA.

    Techniques: Activation Assay, Expressing, Western Blot