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: The appropriate amount of human VEGFR1 phycoerythrin (PE)-conjugated antibody (R&D Systems, FAB321P), VEGFR2 PE-conjugated antibody (BioLegend, 359904), VEGFR3 PE-conjugated antibody (R&D Systems, FAB3492P), PDGFRα PE-conjugated antibody (R&D Systems, FAB1264P), or PDGFRβ PE-conjugated antibody (R&D Systems, FAB1263P) was added to each sample well except the fluorescence-minus-one (FMO) control.

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: The appropriate amount of human VEGFR1 phycoerythrin (PE)-conjugated antibody (R&D Systems, FAB321P), VEGFR2 PE-conjugated antibody (BioLegend, 359904), VEGFR3 PE-conjugated antibody (R&D Systems, FAB3492P), PDGFRα PE-conjugated antibody (R&D Systems, FAB1264P), or PDGFRβ PE-conjugated antibody (R&D Systems, FAB1263P) was added to each sample well except the fluorescence-minus-one (FMO) control.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer immunology research

Article Title: Secretion of a VEGF-blocking scFv enhances CAR T-cell potency

doi: 10.1158/2326-6066.CIR-24-0876

Figure Lengend Snippet: A, Single cell RNA sequencing data derived from[ 29 ] showing expression of KDR (VEGFR2), FLT1 (VEGFR1), VEGFA , NRP1, and NRP2 in CAR T cells from patients that received Tisa-cel and Axi-cel at baseline (prior to CAR T cell production), pre-infusion (post CAR T-cell production), and day 7 after CAR-T cell infusion into the patient (separated into CAR + and CAR − populations). Only cell populations with at least 200 cells in a given patient were included, yielding n=133 measurements taken from N=32 patients, including N=20 baseline, N=31 infusion product, N=29 Day +7 CAR-negative, and N=22 Day +7 CAR-positive sorted PBMC samples. B, Comparison of VEGF signaling family members among Tisa-cel (4–1BB) and Axi-cel (CD28z) patient pre-infusion products. C, Expression of VEGFR1, VEGFR2, NRP1, and NRP2 at baseline or in the infusion product (IP) or monocytes from responding and non-responding patients that received Tisa-cel or Axi-cel. P-values represent a wilcoxon ranksum test, the q-values are FDR-corrected by Benjamini-Hochberg. D, Mean fluorescence intensity (MFI) of VEGFR1 expression in meso-targeting CD8 + CAR + T cells (or untransduced T cells, UTD) with (+) and without (−) stimulation with MESO + K562s for 96 hours and representative histograms. Symbols represent technical triplicates from 2 normal donors (NDs), bars represent mean±SEM, p values by unpaired t-test). E, MFI of VEGFR1 expression in CD70 targeting CD8 + CAR + T cells after activation with CD70 + K562s for 96 hours and representative histograms. Symbols represent technical triplicates from 3 NDs, bars represent mean±SEM, p-value by unpaired t-test) F, VEGF concentration in culture supernatant following mesothelin CAR T-cell production measured by ELISA. (Symbols represent technical triplicates from 2 NDs, bars represent mean±SEM, p-value by one-way ANOVA). IP-infusion product

Article Snippet: The following antibodies were used: anti-mouse IgG (Cell Signaling Technology 4410S) VEGFR1 (R&D systems, FAB321V-100ug), Mouse IgG1k Isotype (R&D systems-IC002V), CD69 (Biolegend, 310918), CD8 (BD-560179, BD-647458), CD4 (BD-651850), CD3 (641406), CD71 (Biolegend-334104), PD1(BD Horizon-563789), TIM3 (BD Horizon-565566), LAG3 (BD Pharmingen-565716), CCR7 (BD Pharmingen-561271), CD45RA (Biolegend-304126), CD45 (Biolegend-368562), TOX (Miltenyi Biotec, 130-118-335), Human IgG1 isotype (Miltenyi Biotec, 130-120-709) .

Techniques: RNA Sequencing, Derivative Assay, Expressing, Comparison, Fluorescence, Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Cancer Research

Article Title: Down-regulation of Vascular Endothelial Growth Factor Receptor 2 Is a Major Molecular Determinant of Proteasome Inhibitor–Mediated Antiangiogenic Action in Endothelial Cells

doi: 10.1158/0008-5472.can-08-3150

Figure Lengend Snippet: Figure 1. Effects of proteasome inhibitors on the VEGFR2 protein expression in HUVECs. Representative Western blot analyses of endothelial cells that were left untreated (solvent only) or treated with lactacystin (1 Amol/L), MG132 (1 Amol/L), and ALLN (10 Amol/L) in a time-dependent manner (A) as indicated and lactacystin, MG132, and ALLN in a concentration- dependent manner (B) as indicated. Total cellular protein was separated by 8% SDS-PAGE. VEGFR2, VEGFR1, tie-2, and tubulin proteins were detected by enhanced chemiluminescence. Comparable results were obtained from three independent experiments. C, effects of MG132 and ALLN on the soluble VEGFR2 expression in supernatants of HUVEC. Soluble VEGFR2 protein content was assayed in culture supernatants by sVEGFR2 ELISA (R&D Systems) according to the manufacturer’s instructions. HUVECs were left untreated (solvent only, DMSO 0.1%) or treated with MG132 (at 1 mmol/L) and ALLN (at 10 Amol/L). Columns, mean values from triplicate experiments; bars, SE (Student’s t test). n.s., not significant.

Article Snippet: The membranes were incubated with the indicated primary antibodies [VEGFR1 (MAB321, clone49560), VEGFR2 (MAB3573, clone89109), and tie-2 (MAB313, clone83711) from R&D Systems; Sp1 (clone PEP2) and flt-1 (clone H-225) from Santa Cruz; tubulin antibody from LabVision], followed by incubation with horseradish peroxidase–conjugated secondary antibodies (anti-mouse or anti-rabbit IgG, Amersham; anti-goat, Dako).

Techniques: Expressing, Western Blot, Solvent, Concentration Assay, SDS Page, Enzyme-linked Immunosorbent Assay