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R&D Systems anti vegfr2
Anti Vegfr2, supplied by R&D Systems, used in various techniques. Bioz Stars score: 94/100, based on 176 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+vegfr2/Mouse+VEGFR2%2FKDR%2FFlk-1+Antibody/pmc13114484-37-80-82
Average 94 stars, based on 176 article reviews

anti vegfr2 - by Bioz Stars, 2026-08

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(A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) using anti-endoglin or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A) , Wild-type (WT) and KI mice HTRZ for either PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were infused with vehicle (0.2% BSA in PBS) or VEGF-A (a total of 3.5μg in 100μl of vehicle) through the carotid artery for 15 days using a mini osmotic pump as in Methods. Left: Brain coronal sections (40μm thick) were prepared and immunostained with anti-Col IV antibodies to visualize brain vessels. Enhanced visualization surfaces were generated using Imaris software from representative confocal images of ipsilateral hemispheres. Scale bar: 50μm. Right: Graph shows total vessel length density in WT and PS1 FAD brains quantified using Imaris 9.9 software as in Methods. (B) , WT and HTRZ for PS1 FAD mutants M146V or I213T mice were injected through the carotid artery for 20 minutes with either vehicle or 100ng of VEGF-A in vehicle prepared as in 1A using a catheter as described in Methods. Brain microvessels (MV) were isolated as in Methods, lysed in Triton X-100 buffer, and subjected to immunoprecipitation (IP) with anti-VEGFR2 antibody or control IgG. Left: IPs were analyzed on Western blots (WBs) using anti-endoglin or anti-VEGFR2 antibodies (upper panel). Input samples are shown in lower panel. β-actin: loading control. Right: Graph shows quantification of endoglin co-IPed with VEGFR2, normalized to IPed VEGFR2. (C) , WT mice were infused for 15 days through the carotid artery with vehicle or VEGF-A in vehicle as in 1A using a mini osmotic pump (as in 1A). For RO injection, mice were treated with vehicle (2% DMSO, 30% PEG 300, 5% Tween-80 in ddH2O) or RO in vehicle (5mg/kg body weight) via five injections in tail vein one injection every three days, with first injection administered 1 hour before osmotic pump implantation. Brain coronal sections (40μm) were prepared and immunostained with anti-Col IV antibodies as in 1A. Left: Representative confocal images of ipsilateral hemispheres are shown prepared as in 1A. Scale bar: 50μm. Right: Graph shows total vessel length density quantified using Imaris software as in 1A. (D) , WT adult mice were treated with either 50μl vehicle as in 1C or 1mg/kg RO in vehicle via carotid artery as in Methods. 15-16 hrs later, 50 μl vehicle prepared as in 1A or 100ng VEGF-A in vehicle was administered via carotid artery for 10-20 minutes using a catheter as in 1B. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. Vinculin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (E) , WT mice and mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T) were treated with vehicle or VEGF-A via carotid artery for 10-20 minutes using a catheter as in 1D. Brain MVs were isolated and extracted as in 1B. Left: p-VEGFR2 (Tyr1054/Tyr1059), VEGFR2, p-ERK1/2 and ERK1/2 are detected on WBs of extracts with specific antibodies in MV extracts. β-actin: loading control. Right: Graphs show fold change of phosphorylated to total protein ratio. (F) , WT pCECs were prepared and treated as in Methods with vehicle (DMSO) or RO (200nM in DMSO) and then stimulated with either vehicle (PBS) or VEGF-A (20ng in PBS) for 15min. Upper: Cells were co-immunostained with either anti-VEGFR2 antibodies (green) or early endosome marker Rab5 (red) and cell nuclei were stained with Hoechst (blue) as in Methods. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in RO-treated WT cells compared to vehicle-treated cells measured with Imaris software. (G) , pCECs from either WT or mice HTRZ for PS1 FAD mutant M146V (WT/M146V) or I213T (WT/I213T), were stimulated with vehicle or VEGF-A in vehicle as in 1F. Upper: Cells were co-stained with anti-VEGFR2 antibodies and early endosome marker Rab5 as in 1F. Cell nuclei were stained with Hoechst (blue) as in 1F. Yellow fluorescence in merged images indicates co-localization of VEGFR2 with Rab5. Scale bar 0.5μm. Lower: Graph shows percent of VEGFR2 co-localized with Rab5 in PS1 FAD WT/M146V or WT/I213T HTRZ mice compared to WT measured with Imaris software. For Figs A-G, data are shown as Mean ± S.E. from at least three independent experiments or as indicated in the dot plots. Statistical analysis was performed using two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Mouse monoclonal anti-Flag tag (M2; F1804) was from Millipore Sigma, anti-GAPDH (2118S) from Cell Signaling Technologies (Beverly, MA), anti-VEGFR2 (OTI12C1) from Origene, anti-endoglin (CD-105; NBP2-22122) and anti-LAMP2 (NBP2-22217) from Novus Biologicals, Inc, anti-Rab5 (D-11) and anti-Rab7 (B-3) from Santa Cruz Biotechnology, Inc. Chicken polyclonal anti-GFAP (ab4674) was from Abcam.

Techniques: Mutagenesis, Generated, Software, Injection, Isolation, Immunoprecipitation, Control, Western Blot, Marker, Staining, Fluorescence

$(A), HEK293T cells transfected with either pCMV3 vector or VEGFR2-Myc-expressing vector were treated with either vehicle or RO overnight as in 1F. Membrane fractions were prepared as in Methods and incubated with vehicle (-) or VEGF-A as in 1F for 30 minutes in the presence of lactacystin. VEGFR2-Myc, VCTF1-Myc, and VCTF2-Myc, were then detected on WB using anti-Myc antibodies. Representative blot shows Myc-labelled fragments as indicated in Figure. (B), Left: HEK293T cells were transfected with VEGFR2-Myc and treated with vehicle or RO as in 3A and extracted in SDS buffer as in methods. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts on WBs with antibodies recognizing the cytoplasmic sequence of VEGFR2 (ab39256). β-actin: loading control. Right: HEK293T cells were transfected as in 3A. Cells were pretreated with 200nm ADAM17 inhibitor D1 (A12: ADAM17 inh) for 1 hour and then stimulated with vehicle or VEGF-A as in 3A for 1h. Cells were extracted as in 3B Left. VEGFR2-Myc and its proteolytic product VCTF1-Myc are detected with anti-Myc antibody on WB as in 3A. Vinculin: loading control. (C), Left: WT pCECs were treated with either vehicle (DMSO; Veh) or RO as in 1F for 15-16 h and extracted in SDS buffer. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from adult WT mice as in 1B following 15 hours of treatment with either vehicle or RO as in 1D and extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. (D), Left: pCECs isolated from WT or PS1 knockout (PS1 KO) mouse embryos were extracted as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: HEK293T cells expressing VEGFR2-Myc were transfected with anti-PS1 siRNA or non-targeting control siRNA (NT siRNA) as in Methods and extracted as in 3B. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts with anti-Myc antibody as in 3A. PS1 N-terminal fragment (PS1/NTF) was detected in cell extracts with anti-PS1 antibody (R222; 41). β-actin: loading control. (E), Left: Extracts from WT and either WT/M146V- or WT/I213T-expressing pCECs were prepared as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from WT and either WT/M146V- or WT/I213T-expressing mice as in 1B and extracted as in 3C Right. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Each WB is representative of at least three independent experiments.$

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells transfected with either pCMV3 vector or VEGFR2-Myc-expressing vector were treated with either vehicle or RO overnight as in 1F. Membrane fractions were prepared as in Methods and incubated with vehicle (-) or VEGF-A as in 1F for 30 minutes in the presence of lactacystin. VEGFR2-Myc, VCTF1-Myc, and VCTF2-Myc, were then detected on WB using anti-Myc antibodies. Representative blot shows Myc-labelled fragments as indicated in Figure. (B), Left: HEK293T cells were transfected with VEGFR2-Myc and treated with vehicle or RO as in 3A and extracted in SDS buffer as in methods. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts on WBs with antibodies recognizing the cytoplasmic sequence of VEGFR2 (ab39256). β-actin: loading control. Right: HEK293T cells were transfected as in 3A. Cells were pretreated with 200nm ADAM17 inhibitor D1 (A12: ADAM17 inh) for 1 hour and then stimulated with vehicle or VEGF-A as in 3A for 1h. Cells were extracted as in 3B Left. VEGFR2-Myc and its proteolytic product VCTF1-Myc are detected with anti-Myc antibody on WB as in 3A. Vinculin: loading control. (C), Left: WT pCECs were treated with either vehicle (DMSO; Veh) or RO as in 1F for 15-16 h and extracted in SDS buffer. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from adult WT mice as in 1B following 15 hours of treatment with either vehicle or RO as in 1D and extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. (D), Left: pCECs isolated from WT or PS1 knockout (PS1 KO) mouse embryos were extracted as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: HEK293T cells expressing VEGFR2-Myc were transfected with anti-PS1 siRNA or non-targeting control siRNA (NT siRNA) as in Methods and extracted as in 3B. VEGFR2-Myc and VCTF1-Myc were detected in cell extracts with anti-Myc antibody as in 3A. PS1 N-terminal fragment (PS1/NTF) was detected in cell extracts with anti-PS1 antibody (R222; 41). β-actin: loading control. (E), Left: Extracts from WT and either WT/M146V- or WT/I213T-expressing pCECs were prepared as in 3C. VEGFR2 and VCTF1 were detected in cell extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Right: Brain MVs were isolated from WT and either WT/M146V- or WT/I213T-expressing mice as in 1B and extracted as in 3C Right. VEGFR2 and VCTF1 were detected in MV extracts on WB with anti-VEGFR2 antibody as in 3B Left. β-actin: loading control. Each WB is representative of at least three independent experiments.

Techniques: Transfection, Plasmid Preparation, Expressing, Membrane, Incubation, Sequencing, Control, Isolation, Knock-Out

(A), HEK293T cells were transfected with VEGFR2-Myc as in 3A and treated with vehicle or VEGF-A for the indicated times, in the presence or absence of RO as in 1F and extracted in SDS buffer. Left: VEGFR2 dimer and monomer were detected on WB with anti-Myc antibodies. β-actin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (B), WT mice were treated with vehicle or RO as in 1D. Mice were treated with VEGF-A, and brain MVs were isolated and extracted as in 1B. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody D5B1. Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (C), WT and WT/M146V or WT/I213T mice were injected with either vehicle or VEGF-A via the carotid artery as in 1D, and brain MVs were prepared as in 1B and extracted as in 3C Right. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody as in 4B Left. β-actin: loading control. Right: graph shows the fold change in the VEGFR2 dimer to monomer ratio. (D), HEK293T cells were co-transfected with VEGFR2-Myc as in 3A and either WT PS1 or PS1 mutant M146V or I213T in FCbAIGW vector as indicated in Figure. Cells were treated with VEGF-A as in 1G for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers were detected in cell extract on WB with anti-Myc antibody as in 4A. Full length PS1 (FL-PS1) and PS1/NTF were detected with R222 (middle). Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (E), HEK293 cells were co-transfected with VEGFR2-Myc and either non-targeting or anti-PS1 siRNA as in 3D. Cells were treated with vehicle (0 lanes) or VEGF-A as in 4D above for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers and VCTF1 were detected on WB with anti-Myc antibody as in 4A. PS1/NTF was detected in cell extracts with R222. β-actin: loading control. Right: graphs show fold change in PS1/NTF levels (upper) and fold change in the VEGFR2 dimer/monomer ratio (lower) following treatment with anti-PS1 siRNA. PS1 downregulation resulted in decreased VEGFR2 dimerization and increased VCTF1-Myc (upper panel). (F), Embryonic brain (E15.5) extract from WT or PS1 knockout (PS1 KO) mice were prepared as described . Representative WB of extracts shows VEGFR2 dimers, detected with anti-VEGFR2 antibody D5B1, and PS1-NTF detected with R222 antibody. Vinculin: loading control. A–E: data are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells were transfected with VEGFR2-Myc as in 3A and treated with vehicle or VEGF-A for the indicated times, in the presence or absence of RO as in 1F and extracted in SDS buffer. Left: VEGFR2 dimer and monomer were detected on WB with anti-Myc antibodies. β-actin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (B), WT mice were treated with vehicle or RO as in 1D. Mice were treated with VEGF-A, and brain MVs were isolated and extracted as in 1B. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody D5B1. Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (C), WT and WT/M146V or WT/I213T mice were injected with either vehicle or VEGF-A via the carotid artery as in 1D, and brain MVs were prepared as in 1B and extracted as in 3C Right. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody as in 4B Left. β-actin: loading control. Right: graph shows the fold change in the VEGFR2 dimer to monomer ratio. (D), HEK293T cells were co-transfected with VEGFR2-Myc as in 3A and either WT PS1 or PS1 mutant M146V or I213T in FCbAIGW vector as indicated in Figure. Cells were treated with VEGF-A as in 1G for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers were detected in cell extract on WB with anti-Myc antibody as in 4A. Full length PS1 (FL-PS1) and PS1/NTF were detected with R222 (middle). Vinculin: loading control. Right: Graph shows the fold change in the VEGFR2 dimer to monomer ratio. (E), HEK293 cells were co-transfected with VEGFR2-Myc and either non-targeting or anti-PS1 siRNA as in 3D. Cells were treated with vehicle (0 lanes) or VEGF-A as in 4D above for the indicated times and extracted in SDS buffer. Left: VEGFR2 dimers and monomers and VCTF1 were detected on WB with anti-Myc antibody as in 4A. PS1/NTF was detected in cell extracts with R222. β-actin: loading control. Right: graphs show fold change in PS1/NTF levels (upper) and fold change in the VEGFR2 dimer/monomer ratio (lower) following treatment with anti-PS1 siRNA. PS1 downregulation resulted in decreased VEGFR2 dimerization and increased VCTF1-Myc (upper panel). (F), Embryonic brain (E15.5) extract from WT or PS1 knockout (PS1 KO) mice were prepared as described . Representative WB of extracts shows VEGFR2 dimers, detected with anti-VEGFR2 antibody D5B1, and PS1-NTF detected with R222 antibody. Vinculin: loading control. A–E: data are presented as mean ± SE from at least three independent experiments. Statistical analysis was performed by two-way ANOVA followed by Tukey post-hoc test. ns = not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Techniques: Transfection, Control, Isolation, Injection, Mutagenesis, Plasmid Preparation, Knock-Out

(A), HEK293T cells were co-transfected with both constructs expressing complementary VEGFR2-Venus fusion proteins as in Methods. Cells were treated with vehicle or VEGF-A for 30 minutes in the presence or absence of RO as in 1F and imaged as in Methods. Left upper panel: Green fluorescence indicates VEGFR2 homodimer formation. Scale bar: 200μm. Left lower panel: Corresponding phase contrast images of cell cultures. Right: Graph shows fold induction of VEGFR2 homodimer formation based on fluorescent intensity measured using ImageJ as in Methods. (B), HEK293T cells were co-transfected with VEGFR2-Myc in pCMV3 vector and either WT PS1 or one of the PS1 FAD mutants indicated in Figure in FCbAIGW. Cells were treated with vehicle or VEGF-A for 0, 15 or 30 mins and then extracted in SDS buffer as in Methods. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody (OTI12C1). PS1 (FL-PS1) and PS1/NTF were detected with R222. Vinculin: loading control. Right: Graph shows fold change in the VEGFR2 dimer to monomer ratio. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was used. ns = not significant, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells were co-transfected with both constructs expressing complementary VEGFR2-Venus fusion proteins as in Methods. Cells were treated with vehicle or VEGF-A for 30 minutes in the presence or absence of RO as in 1F and imaged as in Methods. Left upper panel: Green fluorescence indicates VEGFR2 homodimer formation. Scale bar: 200μm. Left lower panel: Corresponding phase contrast images of cell cultures. Right: Graph shows fold induction of VEGFR2 homodimer formation based on fluorescent intensity measured using ImageJ as in Methods. (B), HEK293T cells were co-transfected with VEGFR2-Myc in pCMV3 vector and either WT PS1 or one of the PS1 FAD mutants indicated in Figure in FCbAIGW. Cells were treated with vehicle or VEGF-A for 0, 15 or 30 mins and then extracted in SDS buffer as in Methods. Left: VEGFR2 dimers and monomers were detected on WB with anti-VEGFR2 antibody (OTI12C1). PS1 (FL-PS1) and PS1/NTF were detected with R222. Vinculin: loading control. Right: Graph shows fold change in the VEGFR2 dimer to monomer ratio. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was used. ns = not significant, **p<0.01, ***p<0.001.

Techniques: Transfection, Construct, Expressing, Fluorescence, Plasmid Preparation, Control

(A), pCECs from WT or HTRZ (WT/M146V or WT/I213T) or HMZG (M146V/M146V or I213T/I213T) mice were extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected on WBs of cell extracts as in 3B with the anti-VEGFR2 antibody (ab39256). β-actin: loading control. (B), Brain MVs were isolated from adult WT mice or mice HTRZ or HMZG for PS1 FAD mutants M146V or I213T (see Suppl. 5A). VEGFR2 and VCTF1 were detected in MV extracts on WB as in Suppl. 5A with anti-VEGFR2 antibody (ab39256). β-actin: loading control.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), pCECs from WT or HTRZ (WT/M146V or WT/I213T) or HMZG (M146V/M146V or I213T/I213T) mice were extracted in SDS buffer as in Methods. VEGFR2 and VCTF1 were detected on WBs of cell extracts as in 3B with the anti-VEGFR2 antibody (ab39256). β-actin: loading control. (B), Brain MVs were isolated from adult WT mice or mice HTRZ or HMZG for PS1 FAD mutants M146V or I213T (see Suppl. 5A). VEGFR2 and VCTF1 were detected in MV extracts on WB as in Suppl. 5A with anti-VEGFR2 antibody (ab39256). β-actin: loading control.

Techniques: Control, Isolation

(A), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and plasmids expressing EphB2/CTF1-Flag, VCTF1-Flag, or vector alone. Cells were stimulated with vehicle (-) or VEGF-A (+) as in 1F and then extracted in SDS buffer. Upper panel: VEGFR2 monomers and dimers are detected on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag and EphB2/CTF1-Flag expression was detected using anti-Flag antibody. Lower panel: GAPDH, loading control. Graph shows fold change of the VEGFR2 dimer to monomer ratio. (B), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and either vector alone or increasing amounts of vector expressing VCTF1-Flag (200ng, 400ng, 800ng, or 1000ng). Forty-eight hours post-transfection cells were stimulated with either vehicle or VEGF-A as in 1F for 20 min and extracted in SDS buffer. Upper panel: VEGFR2 dimers and monomers (VEGFR2-Myc) are detected in cell lysates on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag is detected with anti-Flag antibody. Lower panel: GAPDH: loading control. Bottom: Non-linear regression analysis (inhibitor vs. response, three-parameter model) showed a good fit to the curve (R² = 0.7410), indicating that increase of VCTF1-Flag expression inhibits VEGF-A-induced VEGFR2 dimerization. The red line (squares) represents VEGF-A-treated cells, whereas the blue line (circles) represents vehicle-treated cells. (C), Cells described in 5A were lysed in Triton X-100 buffer as in Methods and lysates were IPed with anti-Flag antibody. Upper panel: VEGFR2-Myc co-IPed with VCTF1-Flag is detected on WB using anti-Myc antibody as in 3A. Second panel: IPed VCTF1-Flag and EphB2/CTF1-Flag are detected on WBs with anti-Flag antibody. Third panel: Input of VEGFR2-Myc is detected with anti-Myc antibody as in 3A. Fourth panel: Input VCTF1-Flag and EphB2/CTF1-Flag are detected with Flag antibody. GAPDH: loading control. (D), bEnd3 cells were transduced with lentiviral vector FCbAIGW expressing VCTF1-Flag or empty vector as in Methods. Cells were treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 7 minutes and extracted in SDS buffer as in Methods. Left: VEGFR2 dimers, monomers, p-VEGFR2 (Tyr1175), p-ERK1/2, ERK1/2 and VCTF1-Flag are detected on WBs with specific antibodies. Right: Graphs show fold change of VEGFR2 dimer to monomer ratio or p-VEGFR2/VEGFR2 and p-ERK1/2/ERK1 protein ratios. (E), bend3 cells expressing either empty vector (FCbAIGW) or VCTF1-Flag as in 5D were seeded as in Methods and treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 6 hours. Upper: Representative photomicrographs show tube-like (loop/mesh) structures. Fluorescent images (EGFP, green) are shown. Scale bar 200μm. Lower: Graph shows quantification of tube formation as average number of loops/meshes per field. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was performed. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and plasmids expressing EphB2/CTF1-Flag, VCTF1-Flag, or vector alone. Cells were stimulated with vehicle (-) or VEGF-A (+) as in 1F and then extracted in SDS buffer. Upper panel: VEGFR2 monomers and dimers are detected on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag and EphB2/CTF1-Flag expression was detected using anti-Flag antibody. Lower panel: GAPDH, loading control. Graph shows fold change of the VEGFR2 dimer to monomer ratio. (B), HEK293T cells were co-transfected with vector expressing VEGFR2-Myc and either vector alone or increasing amounts of vector expressing VCTF1-Flag (200ng, 400ng, 800ng, or 1000ng). Forty-eight hours post-transfection cells were stimulated with either vehicle or VEGF-A as in 1F for 20 min and extracted in SDS buffer. Upper panel: VEGFR2 dimers and monomers (VEGFR2-Myc) are detected in cell lysates on WBs using anti-VEGFR2 antibody OTI12C1. Middle panel: VCTF1-Flag is detected with anti-Flag antibody. Lower panel: GAPDH: loading control. Bottom: Non-linear regression analysis (inhibitor vs. response, three-parameter model) showed a good fit to the curve (R² = 0.7410), indicating that increase of VCTF1-Flag expression inhibits VEGF-A-induced VEGFR2 dimerization. The red line (squares) represents VEGF-A-treated cells, whereas the blue line (circles) represents vehicle-treated cells. (C), Cells described in 5A were lysed in Triton X-100 buffer as in Methods and lysates were IPed with anti-Flag antibody. Upper panel: VEGFR2-Myc co-IPed with VCTF1-Flag is detected on WB using anti-Myc antibody as in 3A. Second panel: IPed VCTF1-Flag and EphB2/CTF1-Flag are detected on WBs with anti-Flag antibody. Third panel: Input of VEGFR2-Myc is detected with anti-Myc antibody as in 3A. Fourth panel: Input VCTF1-Flag and EphB2/CTF1-Flag are detected with Flag antibody. GAPDH: loading control. (D), bEnd3 cells were transduced with lentiviral vector FCbAIGW expressing VCTF1-Flag or empty vector as in Methods. Cells were treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 7 minutes and extracted in SDS buffer as in Methods. Left: VEGFR2 dimers, monomers, p-VEGFR2 (Tyr1175), p-ERK1/2, ERK1/2 and VCTF1-Flag are detected on WBs with specific antibodies. Right: Graphs show fold change of VEGFR2 dimer to monomer ratio or p-VEGFR2/VEGFR2 and p-ERK1/2/ERK1 protein ratios. (E), bend3 cells expressing either empty vector (FCbAIGW) or VCTF1-Flag as in 5D were seeded as in Methods and treated with vehicle (-) or VEGF-A (+) as in Suppl. 4A for 6 hours. Upper: Representative photomicrographs show tube-like (loop/mesh) structures. Fluorescent images (EGFP, green) are shown. Scale bar 200μm. Lower: Graph shows quantification of tube formation as average number of loops/meshes per field. For statistical analysis, two-way ANOVA followed by Tukey post-hoc test was performed. ns = not significant, *p<0.05, **p<0.01, ***p<0.001.

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Transduction

(A), Brain tissue extracts were prepared as in Methods from twelve PS1 FAD patients each carrying a different PS1 mutation, and twelve non-demented controls described in Methods. Left: VEGFR2 dimers and monomers were detected in brain extracts on WBs using anti-VEGFR2 antibody D5B1. Representative gels with control (C1-4) or FAD samples (FAD1-4) expressing mutants P264L, A260V, N135S and P242H respectively are shown. Vinculin: loading control. Right: Graph shows the fold change in VEGFR2 dimer to monomer ratio of FAD and control samples. (B), Brain tissue extract from control and PS1 FAD patient brains described in 7A were prepared and IPed with anti-endoglin antibody (ab252345) or IgG as in Methods. Upper panel: VEGFR2 co-IPed with endoglin was detected on WBs using an anti-VEGFR2 antibody as in 7A. Lower panel: Input samples are shown. Representative gel with control samples (C1, C2) and FAD samples (FAD1, FAD2) expressing mutants A260V and P264L respectively is shown. β-actin: loading control. Right: Graph shows relative levels of VEGFR2 co-precipitated with endoglin. (C), Brain sections from control and PS1 FAD patients were prepared as in Methods and stained for Col IV as in 1A. Upper: Representative images show brain vessels in either PS1 FAD or control (CT) brain sections. Scale bar: 80μm. Lower: Graph shows total vessel length density in PS1 FAD and CT brains quantified with Imaris software as in 1A. A-C , bars represent Mean ± S.E. For statistical analysis, unpaired t-test was performed. *p < 0.05, **p<0.01 and ***p<0.001.

Journal: bioRxiv

Article Title: PS FAD mutants and γ-secretase inhibition accumulate VEGFR2-derived peptide VCTF1 suppressing brain VEGFR2 dimerization, angiogenesis and neuroprotection

doi: 10.64898/2026.05.12.724648

Figure Lengend Snippet: (A), Brain tissue extracts were prepared as in Methods from twelve PS1 FAD patients each carrying a different PS1 mutation, and twelve non-demented controls described in Methods. Left: VEGFR2 dimers and monomers were detected in brain extracts on WBs using anti-VEGFR2 antibody D5B1. Representative gels with control (C1-4) or FAD samples (FAD1-4) expressing mutants P264L, A260V, N135S and P242H respectively are shown. Vinculin: loading control. Right: Graph shows the fold change in VEGFR2 dimer to monomer ratio of FAD and control samples. (B), Brain tissue extract from control and PS1 FAD patient brains described in 7A were prepared and IPed with anti-endoglin antibody (ab252345) or IgG as in Methods. Upper panel: VEGFR2 co-IPed with endoglin was detected on WBs using an anti-VEGFR2 antibody as in 7A. Lower panel: Input samples are shown. Representative gel with control samples (C1, C2) and FAD samples (FAD1, FAD2) expressing mutants A260V and P264L respectively is shown. β-actin: loading control. Right: Graph shows relative levels of VEGFR2 co-precipitated with endoglin. (C), Brain sections from control and PS1 FAD patients were prepared as in Methods and stained for Col IV as in 1A. Upper: Representative images show brain vessels in either PS1 FAD or control (CT) brain sections. Scale bar: 80μm. Lower: Graph shows total vessel length density in PS1 FAD and CT brains quantified with Imaris software as in 1A. A-C , bars represent Mean ± S.E. For statistical analysis, unpaired t-test was performed. *p < 0.05, **p<0.01 and ***p<0.001.

Techniques: Mutagenesis, Control, Expressing, Staining, Software

Mapping of intercellular molecular communication between astrocytes and ECs (A) UMAP visualization of 1,865 astrocytes (ASCs) from sham and ACAS brains (left). Stacked bar plots (right) showing the proportions of the two major subsets, ASC0 and ASC1, in each group. (B) UMAP visualization of subset-specific marker expression. (C) Heatmap displaying the top 12 markers for each subset. (D) Gene Ontology (GO) enrichment analysis based on significant differentially expressed genes (DEGs) using Metascape. (E) Representative immunostaining images of GFAP (red) and Aqp4 (green) in the hippocampal area 35 days after ACAS. (F) Quantification of the numbers of GFAP + and the areas of Aqp4 + astrocytes in the hippocampus of the brain 35 days after ACAS. n = 5–6/group. Student’s t test. Data are shown as mean ± SEM. ∗ p < 0.05. (G) Cell-cell interaction analysis illustrating interactions between EC cells (tip and other ECs) and astrocytes, inferred by the CellChat R package. All predicted ligand-receptor pairs are shown, with dot colors representing interaction probability (red: high, blue: low) and dot sizes indicating the significance level. The VEGF category exhibits a significantly high probability of interactions between ECs and astrocytes in both ACAS and sham. (H) Visualization of signaling pathways in key cell populations. Each segment of the circular diagram corresponds to a cell type, with colored arcs indicating communication between source and target cells. The strength of interactions is represented by the width of the connecting lines, with key signaling molecules (e.g., VEGF, ESAM, and PECAM1) labeled. (I) The heatmap matrix represents the communication probability between source and target cell populations through the VEGF signaling network. The color intensity indicates the interaction strength, with darker shades reflecting higher communication probability. Cell types are color-coded for easy identification. (J) Representative immunostaining images and 3D reconstruction of astrocyte marker GFAP (blue), VEGF (red), tip cell maker CD93 (green), and VEGFR2 (magenta) in the hippocampus 35 days after ACAS. White arrow heads indicate the proximity of VEGFR2 + CD93 + tip cells and VEGF + GFAP + astrocytes. Scale bars are indicated in images.

Journal: iScience

Article Title: Single-cell transcriptomic analyses reveal angiogenic vascular responses to chronic cerebral hypoperfusion

doi: 10.1016/j.isci.2026.115331

Figure Lengend Snippet: Mapping of intercellular molecular communication between astrocytes and ECs (A) UMAP visualization of 1,865 astrocytes (ASCs) from sham and ACAS brains (left). Stacked bar plots (right) showing the proportions of the two major subsets, ASC0 and ASC1, in each group. (B) UMAP visualization of subset-specific marker expression. (C) Heatmap displaying the top 12 markers for each subset. (D) Gene Ontology (GO) enrichment analysis based on significant differentially expressed genes (DEGs) using Metascape. (E) Representative immunostaining images of GFAP (red) and Aqp4 (green) in the hippocampal area 35 days after ACAS. (F) Quantification of the numbers of GFAP + and the areas of Aqp4 + astrocytes in the hippocampus of the brain 35 days after ACAS. n = 5–6/group. Student’s t test. Data are shown as mean ± SEM. ∗ p < 0.05. (G) Cell-cell interaction analysis illustrating interactions between EC cells (tip and other ECs) and astrocytes, inferred by the CellChat R package. All predicted ligand-receptor pairs are shown, with dot colors representing interaction probability (red: high, blue: low) and dot sizes indicating the significance level. The VEGF category exhibits a significantly high probability of interactions between ECs and astrocytes in both ACAS and sham. (H) Visualization of signaling pathways in key cell populations. Each segment of the circular diagram corresponds to a cell type, with colored arcs indicating communication between source and target cells. The strength of interactions is represented by the width of the connecting lines, with key signaling molecules (e.g., VEGF, ESAM, and PECAM1) labeled. (I) The heatmap matrix represents the communication probability between source and target cell populations through the VEGF signaling network. The color intensity indicates the interaction strength, with darker shades reflecting higher communication probability. Cell types are color-coded for easy identification. (J) Representative immunostaining images and 3D reconstruction of astrocyte marker GFAP (blue), VEGF (red), tip cell maker CD93 (green), and VEGFR2 (magenta) in the hippocampus 35 days after ACAS. White arrow heads indicate the proximity of VEGFR2 + CD93 + tip cells and VEGF + GFAP + astrocytes. Scale bars are indicated in images.

Article Snippet: Anti-VEGFR2 Antibody , Cell Signaling Technology , Cat# 2479T; RRID: AB_2212507.

Techniques: Marker, Expressing, Immunostaining, Protein-Protein interactions, Labeling

(A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression of HOXC9 and KDR on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).

Journal: bioRxiv

Article Title: Generation of human hindlimb/genital tubercle progenitors from pluripotent stem cells

doi: 10.64898/2026.04.14.718471

Figure Lengend Snippet: (A) Schematic of the treatment conditions used to test different regimens of RA treatment during hPSC differentiation toward HGTps. (B) Heatmap of differentially expressed pLPM/HGTp, vascular/endothelial markers and posterior HOX genes from bulk RNA-seq on day 10 untreated, late RA and early RA treated cells. Expression values are shown as Z-scores across samples. (C) Immunofluorescence analysis of the expression of HOXC9 and KDR on day 10 untreated, late RA and early RA treated cells. Scale bars represent 100 µm. Image analysis of the percentage of nuclei positive for HOXC9 protein expression is also shown. Graph shows mean values ( n = 3 independent experiments) (* p<0.05, ** p<0.01, *** p<0.001, mean ±SD) (one way ANOVA and Tukey’s multiple comparison test). (D) RNA-seq timecourse analysis of the expression levels of key RA target, genital tubercle mesenchyme, vascular/endothelial, pLPM/HGTp markers. TPM is calculated and analysed using one-way ANOVA and Tukey’s test to calculate adjusted p value (* adj p<0.05, ** adj p<0.01, *** adj p<0.001, mean ±SEM).

Article Snippet: Following primary antibodies were used; TBXT (Abcam, ab209665, 1:1000), ISL1 (DSHB, 39.4D5, 1:200), HAND1 (R&D Systems, AF3168, 1:100), PITX1 (Novus Bio, NBP1-88644, 1:500), HOXC9 (Abcam, ab50839, 1:200), KDR (R&D Systems, AF357, 1:1000), TP63 (Abcam, ab124762, 1:200).

Techniques: RNA Sequencing, Expressing, Immunofluorescence, Comparison

Assembly of an MDM2‐NCL‐PROTAC‐POI quaternary complex. (A) Co‐IP of NCL, MDM2, VEGFR2, and EGFR in U87MG cells using isotype‐matched IgG or an anti‐NCL antibody. (B) Pull‐down assay of NCL, MDM2, VEGFR2, and EGFR in U87MG cells treated with 500 nM biotin‐labelled NC (Bio‐NC) or AS1411 (Bio‐AS1411) for 6 h by streptavidin‐coated magnetic beads. (C) Pull‐down assay of NCL and MDM2 in NCL siRNA (siNCL)‐transfected U87MG cells after treatment with 500 nM Bio‐AS1411 for 6 h. (D) Structure of AS1411‐Ced. (E) Structure of AS1411‐Gef. (F) Absorption spectra of NMM in the presence of 10 µM NC, AS1411, AS1411‐Ced, and AS1411‐Gef. The NMM concentration was 1 µM. (G) Co‐IP of NCL, MDM2, and VEGFR2 in U87MG cells treated with PBS or 500 nM AS1411‐Ced for 6 h using an anti‐VEGFR2 antibody in the presence of 10 µM MG132. (H) Co‐IP of NCL, MDM2, and VEGFR2 in scrambled control siRNA (siCtrl)‐ or siNCL‐transfected U87MG cells after treatment with 500 nM AS1411‐Ced for 6 h using an anti‐VEGFR2 antibody in the presence of 10 µM MG132. (I) Co‐IP of NCL, MDM2, and VEGFR2 in siCtrl or siMDM2‐transfected U87MG cells after treatment with 500 nM AS1411‐Ced for 6 h using an anti‐VEGFR2 antibody in the presence of 10 µM MG132. (J) Co‐IP of NCL, MDM2, and EGFR in U87MG cells treated with PBS or 500 nM AS1411‐Gef for 6 h using an anti‐EGFR antibody in the presence of 10 µM MG132. (K) Co‐IP of NCL, MDM2, and EGFR in siCtrl‐ or siNCL‐transfected U87MG cells after treatment with 500 nM AS1411‐Gef for 6 h using an anti‐EGFR antibody in the presence of 10 µM MG132. (L) Co‐IP of NCL, MDM2, and EGFR in siCtrl‐ or siMDM2‐transfected U87MG cells after treatment with 500 nM AS1411‐Gef for 6 h using an anti‐EGFR antibody in the presence of 10 µM MG132. Experiments were performed in triplicate and repeated three times.

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular Vesicle‐Mediated Nucleolin Transfer in Glioblastoma: A Targetable Axis Driving Blood‐Tumour Barrier Formation

doi: 10.1002/jev2.70268

Figure Lengend Snippet: Assembly of an MDM2‐NCL‐PROTAC‐POI quaternary complex. (A) Co‐IP of NCL, MDM2, VEGFR2, and EGFR in U87MG cells using isotype‐matched IgG or an anti‐NCL antibody. (B) Pull‐down assay of NCL, MDM2, VEGFR2, and EGFR in U87MG cells treated with 500 nM biotin‐labelled NC (Bio‐NC) or AS1411 (Bio‐AS1411) for 6 h by streptavidin‐coated magnetic beads. (C) Pull‐down assay of NCL and MDM2 in NCL siRNA (siNCL)‐transfected U87MG cells after treatment with 500 nM Bio‐AS1411 for 6 h. (D) Structure of AS1411‐Ced. (E) Structure of AS1411‐Gef. (F) Absorption spectra of NMM in the presence of 10 µM NC, AS1411, AS1411‐Ced, and AS1411‐Gef. The NMM concentration was 1 µM. (G) Co‐IP of NCL, MDM2, and VEGFR2 in U87MG cells treated with PBS or 500 nM AS1411‐Ced for 6 h using an anti‐VEGFR2 antibody in the presence of 10 µM MG132. (H) Co‐IP of NCL, MDM2, and VEGFR2 in scrambled control siRNA (siCtrl)‐ or siNCL‐transfected U87MG cells after treatment with 500 nM AS1411‐Ced for 6 h using an anti‐VEGFR2 antibody in the presence of 10 µM MG132. (I) Co‐IP of NCL, MDM2, and VEGFR2 in siCtrl or siMDM2‐transfected U87MG cells after treatment with 500 nM AS1411‐Ced for 6 h using an anti‐VEGFR2 antibody in the presence of 10 µM MG132. (J) Co‐IP of NCL, MDM2, and EGFR in U87MG cells treated with PBS or 500 nM AS1411‐Gef for 6 h using an anti‐EGFR antibody in the presence of 10 µM MG132. (K) Co‐IP of NCL, MDM2, and EGFR in siCtrl‐ or siNCL‐transfected U87MG cells after treatment with 500 nM AS1411‐Gef for 6 h using an anti‐EGFR antibody in the presence of 10 µM MG132. (L) Co‐IP of NCL, MDM2, and EGFR in siCtrl‐ or siMDM2‐transfected U87MG cells after treatment with 500 nM AS1411‐Gef for 6 h using an anti‐EGFR antibody in the presence of 10 µM MG132. Experiments were performed in triplicate and repeated three times.

Article Snippet: The lysate was centrifuged at 13,000 × g at 4°C, and the resulting supernatant was incubated overnight at 4°C with an anti‐EGFR primary antibody (1:50, CST, USA), an anti‐VEGFR2 primary antibody (1:50, CST, USA), or an anti‐NCL primary antibody (1:100, CST, USA).

Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Magnetic Beads, Transfection, Concentration Assay, Control

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