Journal: Cancer research

Article Title: Stromal platelet-derived growth factor receptor-β signaling promotes breast cancer metastasis in the brain

doi: 10.1158/0008-5472.CAN-19-3731

Figure Lengend Snippet: (A) Pdgfb mRNA is increased in DB7 versus Met1 murine mammary cancer cells (bars represent the mean of biological replicates ± SEM, relative to Gapdh). Western blot confirmation of PDGFB protein expression in DB7 and Met1 cells is shown as an inset. (B) Pdgfrb mRNA is elevated in mouse mammary fibroblasts (MMF) compared to DB7 and Met1 murine mammary cancer cells (bars represent the mean of biological replicates ± SEM, relative to Gapdh). Western blot confirmation of PDGFRβ protein expression in MMF is shown as an inset. (C) Expression of Pdgfb mRNA is diminished with lentiviral transduction of individual shRNA constructs (bars represent the mean of biological replicates ± SEM, relative to Gapdh). Western blot of PDGFB in DB7 shCtrl and shPdgfb cells is shown as an inset. (D) Quantification of orthotopic mammary tumor volume over time [n = 5 DB7 shCtrl, n = 5 DB7 shPdgfb-1, shPdgfb-2 and shPdgfb-3; (**) P = 0.0001 at day 30 by one-way ANOVA]. Error bars represent mean ± SD. (E) Kaplan-Meier survival analysis of DB7 shCtrl and DB7 shPdgfb-3 tumor-bearing mice (n = 5 DB7 shCtrl, n = 4 DB7 shPdgfb-3; P = 0.002). Significance determined by log-rank. (F) Representative images of PDGFB immunostaining (red) on orthotopic mammary tumor tissue (blue = DAPI nuclear counterstain). Scale bars = 20μm.

Article Snippet: DB7 cells were transduced with 3 individual shRNA sequences targeting Pdgfb as well as a scrambled control lentivirus (OriGene Technologies Inc.; #TL501604V).

Techniques: Western Blot, Expressing, Transduction, shRNA, Construct, Immunostaining

Journal: Oncotarget

Article Title: Transcriptional co-activator TAZ sustains proliferation and tumorigenicity of neuroblastoma by targeting CTGF and PDGF-β

doi:

Figure Lengend Snippet: (A) BE(2)-C cells were overexpressed with TAZ or overexpressed TAZ cells were knocked down with TAZ siRNA. Total cellular extracts were prepared and subjected to Western blot using antibodies against CTGF and PDGF-β. (B) SK-N-AS cells were knocked down with TAZ siRNA or TAZ siRNA cells were re-introduced with TAZ. Total cellular extracts were prepared and subjected to Western blot using antibodies against CTGF and PDGF-β. GAPDH levels are shown as loading control.

Article Snippet: Mouse anti-TAZ (560235; BD Biosciences), mouse anti-YAP (sc-101199, 1:200), goat anti-CTGF (sc-14939, 1:200) from Santa Cruz company, rabbit anti-PDGF-β (E1A0240-1, 1:1000) from EnoGene, and mouse anti-GAPDH (AG019, 1:1000) from Beyotime, cell cycle regulation antibody sampler kit #9932 from Cell Signaling Technology were used as primary antibodies.

Techniques: Western Blot

Journal: Oncotarget

Article Title: Transcriptional co-activator TAZ sustains proliferation and tumorigenicity of neuroblastoma by targeting CTGF and PDGF-β

doi:

Figure Lengend Snippet: TAZ overexpressing BE(2)-C cells were infected with PDGF-β siRNA. (A) qRT-PCR analysis was performed to determine mRNA expression of PDGF-β. Data represent the means ± SD from three independent experiments. (* P < 0.05). (B) Western blot analysis was performed to determine PDGF-β expression. GAPDH levels are shown as loading control. (C) Cells were seed into a 96-well plate (1000 cells/well), and cell proliferation was determined using cell counting kit-8 assay kit. Data represent the means ± SD from three independent experiments. (* P < 0.05). (D) Colony formation was examined by soft agar assay. (E) Colony number was counted using counter. The values represent the mean ± SD from three independent experiments (* P < 0.05). (F) Cell cycle was analyzed using PI staining and flow cytometry. (G) Immunoblot analysis was performed to determine the expression of G1 cell cycle regulatory proteins including cyclin D1, CDK4 and CDK6. GAPDH levels are shown as loading control.

Article Snippet: Mouse anti-TAZ (560235; BD Biosciences), mouse anti-YAP (sc-101199, 1:200), goat anti-CTGF (sc-14939, 1:200) from Santa Cruz company, rabbit anti-PDGF-β (E1A0240-1, 1:1000) from EnoGene, and mouse anti-GAPDH (AG019, 1:1000) from Beyotime, cell cycle regulation antibody sampler kit #9932 from Cell Signaling Technology were used as primary antibodies.

Techniques: Infection, Quantitative RT-PCR, Expressing, Western Blot, Cell Counting, Soft Agar Assay, Staining, Flow Cytometry

Journal:

Article Title: Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor ? receptor

doi:

Figure Lengend Snippet: Cross-linking analysis of PDGF β receptor in C127 cells. Extracts of PDGF-treated or untreated normal or E5-transformed C127 cells were prepared and treated with the chemical cross-linker BS3 or left untreated. Total PDGF β receptor was immunoprecipitated with αPR and detected by immunoblotting with αPR (A) or anti-phosphotyrosine antibody (B). In B, approximately 3-fold more extract was loaded in lanes 5 and 6 as in the other lanes to facilitate visualization of activated PDGF β receptor species. The position of monomeric mature (m) and precursor (p) PDGF β receptor is shown, as is the position of dimeric receptor (d).

Article Snippet: For specific immunoprecipitation of full-length PDGF β receptor, 8–10 μl of anti-human PDGF β receptor antibody [which we designate αPRex (catalog no. 1263–00, Genzyme)], an mAb recognizing the extracellular ligand-binding domain of human PDGF β receptor, was added to 600–1,000 μg of extract.

Techniques: Transformation Assay, Immunoprecipitation, Western Blot

Journal:

Article Title: Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor ? receptor

doi:

Figure Lengend Snippet: Characterization of Ba/F3 cells. Extracts from Ba/F3 cells expressing the indicated proteins were immunoprecipitated with αPR (A) or the indicated antiserum (B). After gel electrophoresis, PDGF β receptor was detected by immunoblotting with αPR. The position of the mature (m) and precursor (p) forms of the full-length PDGF β receptor is shown, as is the position of the truncated receptor (ΔPR) and phosphorylated truncated receptor (ΔPR-P).

Article Snippet: For specific immunoprecipitation of full-length PDGF β receptor, 8–10 μl of anti-human PDGF β receptor antibody [which we designate αPRex (catalog no. 1263–00, Genzyme)], an mAb recognizing the extracellular ligand-binding domain of human PDGF β receptor, was added to 600–1,000 μg of extract.

Techniques: Expressing, Immunoprecipitation, Nucleic Acid Electrophoresis, Western Blot

Journal:

Article Title: Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor ? receptor

doi:

Figure Lengend Snippet: Oligomerization of the PDGF β receptor in response to the E5 protein. Extracts of Ba/F3 cells expressing the indicated proteins were immunoprecipitated with αPR, which recognizes total PDGF β receptor or with αPRex, which recognizes only the full-length receptor, as indicated. Receptor species immunoprecipitated by these antisera were detected by immunoblotting with αPR. Position of mature (m) and precursor (p) forms of full-length receptor is shown, as is position of truncated receptor (ΔPR).

Article Snippet: For specific immunoprecipitation of full-length PDGF β receptor, 8–10 μl of anti-human PDGF β receptor antibody [which we designate αPRex (catalog no. 1263–00, Genzyme)], an mAb recognizing the extracellular ligand-binding domain of human PDGF β receptor, was added to 600–1,000 μg of extract.

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor ? receptor

doi:

Figure Lengend Snippet: Receptor oligomerization by wild-type and mutant E5 proteins. Ba/F3 cells expressing the full-length and truncated forms of the PDGF β receptor were engineered to express the wild-type E5 protein or a mutant protein containing the Asp-to-Val substitution at position 33 (D33V). Detergent extracts were immunoprecipitated with αPRex and immunoblotted with αPR to detect truncated receptor associated with the full-length receptor. Position of receptor species is indicated as described in the legend to Fig. ​Fig.44.

Article Snippet: For specific immunoprecipitation of full-length PDGF β receptor, 8–10 μl of anti-human PDGF β receptor antibody [which we designate αPRex (catalog no. 1263–00, Genzyme)], an mAb recognizing the extracellular ligand-binding domain of human PDGF β receptor, was added to 600–1,000 μg of extract.

Techniques: Mutagenesis, Expressing, Immunoprecipitation

Journal:

Article Title: Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor ? receptor

doi:

Figure Lengend Snippet: Trans-phosphorylation of kinase-negative PDGF β receptor. Extracts of Ba/F3 cells expressing the indicated proteins were immunoprecipitated with αPR, and tyrosine-phosphorylated species were detected by immunoblotting with anti-phosphotyrosine antibody. Position of receptor species is indicated as described in the legend to Fig. ​Fig.44.

Article Snippet: For specific immunoprecipitation of full-length PDGF β receptor, 8–10 μl of anti-human PDGF β receptor antibody [which we designate αPRex (catalog no. 1263–00, Genzyme)], an mAb recognizing the extracellular ligand-binding domain of human PDGF β receptor, was added to 600–1,000 μg of extract.

Techniques: Expressing, Immunoprecipitation, Western Blot

Journal:

Article Title: Bovine papillomavirus E5 protein induces oligomerization and trans-phosphorylation of the platelet-derived growth factor ? receptor

doi:

Figure Lengend Snippet: Characterization of PDGF β receptor phosphorylation. Extracts of Ba/F3 cells coexpressing the E5 protein, the full-length kinase-inactive receptor, and the truncated kinase-active receptor were immunoprecipitated with αPR, αPRex, or αE5, as indicated. The indicated samples also were treated with protein tyrosine phosphatase (PTPase). After gel electrophoresis, the immunoprecipitated receptor species were detected by probing the filter with anti-phosphotyrosine antibody (A) and αPR (B). The position of receptor species is indicated as described in the legend to Fig. ​Fig.33.

Article Snippet: For specific immunoprecipitation of full-length PDGF β receptor, 8–10 μl of anti-human PDGF β receptor antibody [which we designate αPRex (catalog no. 1263–00, Genzyme)], an mAb recognizing the extracellular ligand-binding domain of human PDGF β receptor, was added to 600–1,000 μg of extract.

Techniques: Immunoprecipitation, Nucleic Acid Electrophoresis