Journal: Circulation

Article Title: Mechanosensitive p27 Kip1 Regulation and Cell Cycle Entry in Vascular Smooth Muscle Cells

doi: 10.1161/01.cir.0000079102.08464.e2

Figure Lengend Snippet: Figure 6. Stretch-induced Akt activation is growth factor and growth factor receptor independent. a, Neutralizing antibodies (nAb) against IGF, bFGF, or PDGF were added, and cells were stretched for 15 minutes. Phosphorylation of Akt was determined by immunoblotting (WB) with phosphospecific antibody. Pan-Akt served as housekeeping protein. b, Growth factor receptor inhibitors AG1478 (EGF), AG1296 (PDGF), and AG1024 (IGF-1) were added, and cells were stretched for 15 minutes or 24 hours to determine Akt activation and p27Kip1 expression by immunoblotting (WB), respectively. Pan-Akt served as housekeeping protein.

Article Snippet: Mouse monoclonal anti-p27Kip1, antiretinoblastoma (RB), anti-Cdk2 (Santa Cruz Biotechnology, Santa Cruz, Calif), anti-basic fibroblast growth factor (bFGF), antiinsulinlike growth factor (IGF), and anti–platelet-derived growth factor (PDGF) (R&D Systems, GMBH, Weisbaden, Germany) were also used.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Expressing

Journal: The Journal of biological chemistry

Article Title: High-affinity binding of basic fibroblast growth factor and platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan.

doi: 10.1074/jbc.274.24.16831

Figure Lengend Snippet: FIG. 1. Binding of bFGF and PDGF-AA to plastic-immobilized NG2 fragments. Increasing concentrations of bFGF (A) or PDGF-AA (B) were added to microtiter wells coated with NG2 frag- ments or BSA at a concentration of 3 mg/ ml. Bound bFGF and PDGF-AA were de- tected with growth factor-specific poly- clonal antibodies. The binding assay was performed as described under “Experi- mental Procedures.” Curves in NG2- coated wells represent the best fit deter- mined by nonlinear regression analysis.

Article Snippet: Goat polyclonal antibodies against human bFGF, EGF, PDGF-AA, PDGF-BB, TGF-b1, and VEGF165 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Concentration Assay

Journal: The Journal of biological chemistry

Article Title: High-affinity binding of basic fibroblast growth factor and platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan.

doi: 10.1074/jbc.274.24.16831

Figure Lengend Snippet: FIG. 2. Inhibition of binding of bFGF or PDGF-AA to immobilized NG2D2 or NG2D3 by soluble NG2 fragments. Microtiter wells coated with NG2/D2 (A) or NG2/D3 (B) were incu- bated with 15 nM FGF (f) or 15 nM PDGF-AA (G) in the presence of increas- ing concentrations of NG2/D2 (A) or NG2/D3 (B), respectively. Bound growth factor was detected with growth factor- specific polyclonal antibodies. The OD at 450 nm obtained with bound growth fac- tors in the absence of soluble NG2 frag- ments was set at 100%. Each point repre- sents the mean 6 S.D. of duplicate values from three separate experiments.

Article Snippet: Goat polyclonal antibodies against human bFGF, EGF, PDGF-AA, PDGF-BB, TGF-b1, and VEGF165 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Inhibition, Binding Assay

Journal: The Journal of biological chemistry

Article Title: High-affinity binding of basic fibroblast growth factor and platelet-derived growth factor-AA to the core protein of the NG2 proteoglycan.

doi: 10.1074/jbc.274.24.16831

Figure Lengend Snippet: FIG. 3. Binding of NG2 fragments to plastic-immobilized growth factors. NG2/EC minus GAGs was diluted at 20 (black columns) and 100 (gray columns) ng/ml and added to microtiter wells coated with bFGF, PDGF-AA, PDGF-BB, TGF-b1, or BSA at a concentration of 2 mg/ml. The inset shows binding of D3 at a concentration of 40 (black columns) or 200 (gray columns) ng/ml to bFGF, PDGF-AA, and BSA-coated wells (2 mg/ml). Bound NG2 fragments were detected with NG2/ EC-specific or NG2/D3-specific polyclonal antibodies. The data represent the mean 6 S.D. of duplicate values from four separate experiments.

Article Snippet: Goat polyclonal antibodies against human bFGF, EGF, PDGF-AA, PDGF-BB, TGF-b1, and VEGF165 were purchased from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Concentration Assay

Journal: Cancer Gene Therapy

Article Title: Knock-out of 5-lipoxygenase in overexpressing tumor cells—consequences on gene expression and cellular function

doi: 10.1038/s41417-022-00531-9

Figure Lengend Snippet: The DEGs were further validated by RT-qPCR and analysis of protein expression. Four differentially regulated genes per cell line are depicted. Gene expression was normalized to ACTB (housekeeping gene) and the corresponding control vector cells (2 −ΔΔct method). A qPCR analysis of four representative DEGs from HCT-116 5-LO KO cells. B Secretion of TGFβ 2 into the cell culture supernatants of HCT-116 5-LO KO cells measured via ELISA. Depicted are the relative TGFβ 2 amounts in % compared to the vector control (mean TGFβ 2 secretion from vector control cells: 231.9 ± 140 pg/µg total protein). C qPCR analysis of four representative DEGs from HT-29 5-LO KO cells and D U-2 OS cells. E Cytokine release from U-2 OS 5-LO KO cells. Data are depicted as relative cytokine amounts in % compared to the corresponding control vector cells. PDGF-AA was assessed by ELISA while fractalkine ( CX3CL1 ) and MCP-1 ( CCL2 ) were measured via FACS employing a cytometric bead array (mean secretion from vector control cells: fractalkine, 64.9 ± 9.2 pg/µg protein; MCP-1, 978.2 ± 101.8 pg/µg protein; PDGF-AA, 255,6 ± 95.6 pg/µg protein). The complete mRNA expression data can be found in supplementary Fig. and supplementary table . All data are presented as mean + SD of three independent experiments. Asterisks indicate significant changes vs. control vector cells. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Article Snippet: PDGF-AA was measured with the DuoSet ® Human PDGF-AA ELISA Kit (R&D systems) following the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Control, Plasmid Preparation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Biology of reproduction

Article Title: Proliferation and differentiation of rat theca-interstitial cells: comparison of effects induced by platelet-derived growth factor and insulin-like growth factor-I.

doi: 10.1095/biolreprod60.3.546

Figure Lengend Snippet: FIG. 2. Effects of PDGF (0.1–1 nM) on DNA synthesis by rat T-I cells in the presence and in the absence of IGF-I (10 nM). The cells were cultured in serum-free medium for 48 h in 96-well plates at a concentration of 35 000 cells/well. Each well contained 0.25 ml of medium. During the last 24 h of culture, [3H]thymidine (1 mCi/well) was added to determine DNA synthesis. Bars indicate the mean of each treatment, and vertical lines indicate the SEM from at least six replicates; means with no superscripts in common are significantly different (p , 0.05).

Article Snippet: Recombinant PDGF-AB heterodimer and anti-human PDGF neutralizing antibody (Ab-PDGF) were purchased from R&D Systems (Minneapolis, MN).

Techniques: DNA Synthesis, Cell Culture, Concentration Assay

Journal: Biology of reproduction

Article Title: Proliferation and differentiation of rat theca-interstitial cells: comparison of effects induced by platelet-derived growth factor and insulin-like growth factor-I.

doi: 10.1095/biolreprod60.3.546

Figure Lengend Snippet: FIG. 3. Effect of Ab-PDGF (10 mg/ml) on DNA synthesis by rat T-I cells. The cells were cultured in serum-free medium with or without PDGF (1 nM), IGF-I (10 nM), and/or Ab-PDGF (10 mg/ml) for 48 h. The cultures were carried out in 96-well plates at a concentration of 35 000 cells/well. Each well contained 0.25 ml of medium. During the last 24 h of culture, [3H]thymidine (1 mCi/well) was added to determine DNA synthesis. Bars indicate the mean of each treatment, and vertical lines indicate the SEM from at least six replicates; * denotes values significantly different from control (p , 0.05).

Article Snippet: Recombinant PDGF-AB heterodimer and anti-human PDGF neutralizing antibody (Ab-PDGF) were purchased from R&D Systems (Minneapolis, MN).

Techniques: DNA Synthesis, Cell Culture, Concentration Assay, Control

Journal: Biology of reproduction

Article Title: Proliferation and differentiation of rat theca-interstitial cells: comparison of effects induced by platelet-derived growth factor and insulin-like growth factor-I.

doi: 10.1095/biolreprod60.3.546

Figure Lengend Snippet: FIG. 4. Effect of PDGF (1 nM) and IGF-I (10 nM) on the number of steroidogenically active [3b-HSD(1)] and steroidogenically inactive [3b- HSD(2)] rat T-I cells. T-I cells were cultured in serum-free medium with or without treatments for 48 h in 24-well plates at a concentration of 350 000 cells per well. Each well contained 1 ml of medium. At the end of the culture period the cells were trypsinized, and steroidogenically active cells were stained by a histochemical reaction identifying 3b-HSD activ- ity. Bars indicate the mean for each treatment, and vertical lines indicate the SEM from four cultures; * denotes values significantly different from control; p , 0.05.

Article Snippet: Recombinant PDGF-AB heterodimer and anti-human PDGF neutralizing antibody (Ab-PDGF) were purchased from R&D Systems (Minneapolis, MN).

Techniques: Cell Culture, Concentration Assay, Staining, Control