rabbit anti pdgfr β antibody  (R&D Systems)

Journal: bioRxiv

Article Title: Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

doi: 10.1101/2025.02.27.640684

Figure Lengend Snippet: The numbers of PDGFRs resulted from nonspecific binding between PDGFR antibodies and PDGFRs. Error bars represent the standard error of mean (SEM). (*p<0.05, **p<0.01, ***p<0.001 as determined by one-way ANOVA and multi-comparison post-hoc Tukey’s test.) The same method was used to calculate all p-values in this study.

Article Snippet: After being briefly vortexed and centrifuged at 12,000× g for 10 min at 4 °, the cell lysates went through the protocol of Human Phospho-VEGFR1/Flt-1 DuoSet IC ELISA kit (R&D Systems, DYC4170), Human Phospho-VEGFR2/KDR DuoSet IC ELISA kit (R&D Systems, DYC1766), or Human Phospho-PDGFRβ DuoSet IC ELISA kit (R&D Systems, DYC1767) to assess the phosphorylation levels of VEGFR1, VEGFR2, or PDGFRβ, respectively.

Techniques: Binding Assay, Comparison

Journal: bioRxiv

Article Title: Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

doi: 10.1101/2025.02.27.640684

Figure Lengend Snippet: HBMECs were starved with low-serum medium for 6 hours and stimulated with growth factors at the indicated concentrations for 30 min (VEGFR1) or 10 min (VEGFR2). The receptor activation was picked up either by ELISA detecting the pan-tyrosine phosphorylation of VEGFR1 (A–C) or VEGFR2 (D) , or by immunoblotting and probed with primary antibody targeting VEGFR2 phosphorylation at tyrosine site 1175 (E) .

Article Snippet: After being briefly vortexed and centrifuged at 12,000× g for 10 min at 4 °, the cell lysates went through the protocol of Human Phospho-VEGFR1/Flt-1 DuoSet IC ELISA kit (R&D Systems, DYC4170), Human Phospho-VEGFR2/KDR DuoSet IC ELISA kit (R&D Systems, DYC1766), or Human Phospho-PDGFRβ DuoSet IC ELISA kit (R&D Systems, DYC1767) to assess the phosphorylation levels of VEGFR1, VEGFR2, or PDGFRβ, respectively.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

doi: 10.1101/2025.02.27.640684

Figure Lengend Snippet: (A– G) HDMEC ( PDGFRA −/− and PDGFRB −/− ) were starved with low-serum medium for 6 hours and stimulated with growth factors at the indicated concentrations for 30 min (VEGFR1) or 10 min (VEGFR2). (A–C) The fold changes of VEGFR1 phosphorylation induced were assessed via ELISA. (D–G) The fold changes of VEGFR1 phosphorylation induced were assessed via immunoblotting. (H) The fold changes of PDGFRβ phosphorylation in HDFs induced by 30-min VEGF-A or PDGF-BB treatment were measured via ELISA.

Article Snippet: After being briefly vortexed and centrifuged at 12,000× g for 10 min at 4 °, the cell lysates went through the protocol of Human Phospho-VEGFR1/Flt-1 DuoSet IC ELISA kit (R&D Systems, DYC4170), Human Phospho-VEGFR2/KDR DuoSet IC ELISA kit (R&D Systems, DYC1766), or Human Phospho-PDGFRβ DuoSet IC ELISA kit (R&D Systems, DYC1767) to assess the phosphorylation levels of VEGFR1, VEGFR2, or PDGFRβ, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: Cross-family Signaling: PDGF Mediates VEGFR Activation and Endothelial Function

doi: 10.1101/2025.02.27.640684

Figure Lengend Snippet: (A and B) Subsequent to a 6-hour period of serum deprivation (1% FBS), HBMECs underwent treatment with growth factors as detailed. Post-incubation, the cell culture supernatant underwent centrifugation and analysis as per the protocols outlined in the Human VEGF ELISA Kit – Quantikine (R&D Systems, SVE00). (C) A fitting curve was generated to depict the correlation between VEGF-A concentration and in-vitro incubation duration. The determination of VEGF-A half-life time was computed utilizing the formula t = − t 1 · ln 2, yielding a value of 26.76 minutes.

Article Snippet: After being briefly vortexed and centrifuged at 12,000× g for 10 min at 4 °, the cell lysates went through the protocol of Human Phospho-VEGFR1/Flt-1 DuoSet IC ELISA kit (R&D Systems, DYC4170), Human Phospho-VEGFR2/KDR DuoSet IC ELISA kit (R&D Systems, DYC1766), or Human Phospho-PDGFRβ DuoSet IC ELISA kit (R&D Systems, DYC1767) to assess the phosphorylation levels of VEGFR1, VEGFR2, or PDGFRβ, respectively.

Techniques: Incubation, Cell Culture, Centrifugation, Enzyme-linked Immunosorbent Assay, Generated, Concentration Assay, In Vitro

Journal: Human Mutation

Article Title: Temperature as a Key Modulator: Investigating Phosphorylation Patterns of p.Asn666 PDGFRB Variants and Their Role in Downstream Signaling

doi: 10.1155/humu/6664372

Figure Lengend Snippet: (a) All PDGFR β substitutions (p.Asn666Lys, p.Asn666Ser, p.Asn666His, and p.Asn666Tyr) increase basal phosphorylation at 37°C. (b) Exposure to 32°C for 6 h further increases phosphorylation in the p.Asn666Tyr, p.Asn666Lys, and p.Asn666His substitutions. Total phosphorylated PDGFR β levels were measured by ELISA. Nontransduced immortalized fibroblasts were referred to as “nt,” while fibroblasts transduced with PDGFRB wild-type vector were denoted as “WT PDGFRB .” (a) One-way ANOVA with Dunnett's test compared wild-type PDGFRB and variants at 37°C (⁣ ∗∗∗∗ p < 0.0001). (b) Cells were incubated at 32°C or 37°C, and two-way ANOVA with Šídák's test compared phosphorylation between temperatures for each variant (⁣ ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001).

Article Snippet: In brief, ELISA analysis was conducted using a DuoSet IC PDGFR β kit (# DYC1767-2, R&D Systems, Minneapolis, MN) following the guidelines provided by the manufacturer.

Techniques: Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Transduction, Plasmid Preparation, Incubation, Variant Assay

Journal: Human Mutation

Article Title: Temperature as a Key Modulator: Investigating Phosphorylation Patterns of p.Asn666 PDGFRB Variants and Their Role in Downstream Signaling

doi: 10.1155/humu/6664372

Figure Lengend Snippet: Effect of reduced temperature (32°C) and ligand stimulation on PDGFR β -specific tyrosine residue phosphorylation. Representative immunoblots showing phosphorylation of PDGFR β at p-Y-740, p-Y-751, p-Y-771, p-Y-1009, and p-Y-1021. At 37°C, p.Asn666Lys and p.Asn666Ser variants exhibited nearly identical signal intensity, whereas p.Asn666His and p.Asn666Tyr showed a distinct pattern. At 32°C, all variants exhibited a variant-specific increase in phosphorylation. PDGF stimulation (10 ng/mL) altered phosphorylation patterns, increasing the upper PDGFR β band, likely representing the fully mature, membrane-associated receptor. Full immunoblots, also overexposed, are shown in Figures and .

Article Snippet: In brief, ELISA analysis was conducted using a DuoSet IC PDGFR β kit (# DYC1767-2, R&D Systems, Minneapolis, MN) following the guidelines provided by the manufacturer.

Techniques: Residue, Phospho-proteomics, Western Blot, Variant Assay, Membrane

Journal: Human Mutation

Article Title: Temperature as a Key Modulator: Investigating Phosphorylation Patterns of p.Asn666 PDGFRB Variants and Their Role in Downstream Signaling

doi: 10.1155/humu/6664372

Figure Lengend Snippet: Effects of lower physiological temperature (32°C) on PDGFR β downstream signaling. (a) Immunoblots showing temperature-dependent changes in p-AKT-Ser473, p-AKT-Thr308, p-STAT1, p-STAT3-Tyr705, p-STAT6, and p-PLC γ 1, in immortalized fibroblasts expressing the four p.Asn666 variants. GAPDH served as a loading control. Full immunoblots are shown in Figure . (b,c) Quantification of downstream signaling is presented as the ratio of phosphorylated protein to GAPDH. (b) Normalized values for each variant compared to the wild type at 37°C. (c) Rations for each variant at 32°C versus 37°C. Symbols represent fold changes: “-” (≥ 30% decrease), “≈” (−30% to +50%), “(+)” (150–200%), “+” (2- to 4-fold), “++” (4- to 10-fold), “+++” (10- to 50-fold), “++++” (> 50-fold). Bar charts summarizing these data are provided in Figures and .

Article Snippet: In brief, ELISA analysis was conducted using a DuoSet IC PDGFR β kit (# DYC1767-2, R&D Systems, Minneapolis, MN) following the guidelines provided by the manufacturer.

Techniques: Western Blot, Expressing, Control, Variant Assay

Journal: Investigative ophthalmology & visual science

Article Title: Corneal Vascularization Associated With a Novel PDGFRB Variant.

doi: 10.1167/iovs.64.14.9

Figure Lengend Snippet: FIGURE 2. (A) Levels of phosphorylated PDGFRβ measured by ELISA analysis. The untreated wild-type and p.(Ser548Tyr) cells have compa- rable PDGFRβ phosphorylation levels. Upon stimulation with PDGF (2.5 ng/mL and 12.5 ng/mL), higher PDGFRβ phosphorylation levels were found in p.(Ser548Tyr) cells. Data are shown as mean ± SEM of three independent experiments. Paired t-test was performed to compare wild-type and p.(Ser548Tyr) response to PDGF stimulation, * P < 0.01. (B) Representative images showing effect of ligand stimulation on PDGFRβ downstream signaling proteins. Increased levels of downstream signaling proteins, p-AKTSer473, p-AKTTyr308, and p-PLCɣ1 upon PDGF stimulation were seen in p.(Ser548Tyr) cells, whereas no such effect was seen on p-STAT1. Upon stimulation with low levels of PDGF, comparable levels of p-MAPK3/ERK1 were found, whereas at high concentration (12.5 ng/mL) increased phosphorylation levels were observed in p.(Ser548Tyr) cells. The p-AKTSer473/GAPDH, p-AKTTyr308/GAPDH, and p-PLC ɣ1/GAPDH ratios upon stimulation with 2.5 ng/mL PDGF were 4.9, 12.8, and 3.7 folds higher in p.(Ser548Tyr) cells compared to wild-type cells. All results were replicated in at least three independent experiments. ELISA analysis data point distributions, full immunoblottings, and quantification of representative images are shown in Supplementary Figures S4 and S5.

Article Snippet: ELISA and immunoblot analyses were performed as previously described.2 In brief, ELISA analysis was conducted using a DuoSet IC PDGFRβ kit (# DYC1767-2, R&D systems) in accordance with the guidelines provided by the manufacturer.

Techniques: Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Concentration Assay

Journal: Investigative ophthalmology & visual science

Article Title: Corneal Vascularization Associated With a Novel PDGFRB Variant.

doi: 10.1167/iovs.64.14.9

Figure Lengend Snippet: FIGURE 4. Effect of tyrosine kinase inhibitors on PDGFRβ and downstream signaling proteins. Panels (A) and (B) show ligand-induced PDGFRβ autophosphorylation levels measured with ELISA in p.(Ser548Tyr) (A) and wild type (B) cells after incubation with four tyro- sine kinase inhibitors (axitinib, dasatinib, imatinib, and sunitinib) at different concentrations. Data are shown as mean ± SEM of three independent experiments. Paired t-test was performed to compare drug effect on PDGF-stimulated PDGFRβ. * P < 0.05, ** P < 0.01, and *** P < 0.001. Panels (C) and (D) show representative images of immunoblot analysis of downstream signaling proteins in p.(Ser548Tyr) (C) and wild-type (D) cells after incubation with tyrosine kinase inhibitors at different concentrations. Axitinib, at all used concentrations, most effectively inhibited phosphorylated PDGF/PDGFRβ and downstream signaling cascades. All results were replicated in at least three independent experiments. ELISA analysis data point distributions, full immunoblottings, and quantification of representative images are shown in Supplementary Figures S9 to S11.

Article Snippet: ELISA and immunoblot analyses were performed as previously described.2 In brief, ELISA analysis was conducted using a DuoSet IC PDGFRβ kit (# DYC1767-2, R&D systems) in accordance with the guidelines provided by the manufacturer.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Western Blot