MA5-13182

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg) of K-562 (Lane 1), Jurkat (Lane 2), MCF-7 (Lane 3) and HeLa (Lane 4). The blots were probed with VEGF Mouse monoclonal Antibody (Product # MA5-13182, 2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A28177, 0.4 µg/mL, 1:2500 dilutions). Isoforms of 15 kDa, 17 kDa, 25 kDa and 35 kDa bands corresponding to VEGF was observed across all cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0341BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-VEGF Monoclonal Antibody (JH121) (Product # MA5-13182) and a 50-52 kDa band corresponding to VEGF was observed across cell lines tested except DU 145 (untreated). Whole cell extracts (30 µg lysate) of DU 145 (Lane 1), DU 145 treated with 1X-PTI for 4 hours, HACAT (Lane 3) and HACAT treated with 0.5 mM H2O2 for 30 minutes followed by 1X-PTI for 4 hours (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Reagent Kit (Product # WP20005). Uncharacterized bands (*) were observed at around 28 kDa and 35 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Vascular Endothelial Growth Factor (VEGF, green) in Hela cells. The cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and blocked with 3% Blocker BSA (Product # 37525) for 30 minutes at room temperature. Cells were stained with (left panel) or without (right panel) a VEGF polyclonal antibody (Product # MA5-13182) at a dilution of 1:40 for at least 1 hour at room temperature, and then incubated with a Dylight 488 goat anti-mouse IgG secondary antibody at a dilution of 1:1000 for 45 minutes at room temperature. F-actin (both panels, red) was stained by Dylight 554 Phalloidin (Product # 21834) and nuclei (both panels, blue) were stained with DAPI (Product # 46190). Images were taken at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of VEGF was performed using DU 145 cells and DU 145 cells treated with 1X-PTI for 4 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with VEGF Monoclonal Antibody (JH121) (Product # MA5-13182) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image of DU 145 PTI-treated cells showing induction of VEGF protein in cytoplasm. Panel e represents the merged image of DU 145 cells, that shows lower expression for VEGF protein. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of VEGF was performed using HACAT cells and HACAT cells treated with 0.5 mM H2O2 for 30 minutes followed by 1X-PTI for 4 hours. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with VEGF Monoclonal Antibody (JH121) (Product # MA5-13182) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing induction of VEGF expression in cytoplasm in treated HACAT cells. Panel e represents untreated HACAT cells showing lower VEGF expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Formalin-fixed, paraffin-embedded human angiosarcoma stained with VEGF antibody using peroxidase-conjugate and AEC chromogen. Note cytoplasmic staining of tumor cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of VEGF showing staining in the cytoplasm of paraffin-embedded human liver tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a VEGF Mouse Monoclonal Antibody (Product # MA5-13182) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of VEGF showing staining in the cytoplasm of paraffin-embedded mouse kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a VEGF Mouse Monoclonal Antibody (Product # MA5-13182) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: FIGURE 5 Immunohistochemical evaluation of angiogenic marker VEGF positive expression. (A) 14 days post-surgery, the positive expression level of VEGF (in green arrows) in the 10 muM Hst1 group was higher. The scale bar = 50 mum. (B) Quantitative analysis of the VEGF expression level at wound sites. 14 days post-surgery, the VEGF expression in the groups of 10 muM Hst1 and ADM + 10 muM Hst1 was significantly higher compared with the control group and ADM group. SS: The 10 muM Hst1 group and 10 muM Hst1 + ADM group vs the control group ( P < 0.05).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 Proliferation and apoptosis. a Immunohistochemical staining for proliferation marker Ki67 in kidney tissue (left), alongside quantification of the percentage of Ki67-positive cells (right). b Western blot on kidney tissue measuring proliferation markers PCNA, VEGF, survivin, and beta-actin (left) alongside quantification (right). c Immunohistochemical staining for apoptosis marker Caspase-3 in kidney tissue. d Western blot on kidney tissue measuring apoptosis markers BAX, Bcl-2, and Caspase-3 (left) alongside quantification (right). Each group has n = 3 mice. Significant difference a p < 0.05: relative to untreated control; b p < 0.05: relative to AKI; c p < 0.05: relative to AKI-EV; d p < 0.05: relative to AKI-EV-pFUS

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 The expression of VEGFA and proliferation/invasion-related downstream protein of alphaT3-1 cells. (A) Schematic diagram of the target protein band. (B) The quantification of VEGFA expressions using western blot analysis. (C) The quantification of MMP2 expressions using western blot analysis. (D) The quantification of MMP9 expressions using western blot analysis. (E) The quantification of Vimentin expressions using western blot analysis. (F) The quantification of Snail expressions using western blot analysis. Data were represented as means +- SD (n = 5). #### p < 0.0001 and ## p < 0.01 vs. miR-134 mimics + PBS group. &&&& p < 0.0001 vs. miR-134 inhibitor + PBS group. **** p < 0.0001 vs. black vector control + PBS group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 7 (a) qRT-PCR analysis of the CD34, VEGF, MMP9, and SDF-1 expressions in each group. (b) Western blot analysis results of CD34, VEGF, MMP9, and SDF-1 protein expressions in each group. * P < 0.05 compared with the blank group.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 5 LTBP4 induces angiogenesis for kidney repair in mice subjected to unilateral ureteral obstruction (UUO). Tubulointerstitial fibrosis was induced by UUO for 5 days in wild-type (WT) and Ltbp4 knockout ( Ltbp4S -/-) mice. A Representative histological images with Masson's trichrome and immunofluorescence staining of kidneys for CD31 after UUO. Scale bars, 100 mum. B Quantitative results of vessels per HPF in the mouse kidneys. C Protein expression of VEGF and CD31 in the kidneys of mice with UUO, as detected by using immunoblotting. GAPDH served as an internal control. D , E Densitometric analyses of immunoblot images for VEGF ( D ) and CD31 ( E ) are shown. Data are presented as the mean +- SEM for each group ( n = 3). * P < 0.05, ** P < 0.01, *** P < 0.001. Results representative of three independent experiments performed in triplicate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 4 Immunohistochemical evaluation of letrozole-treated chorioallantoises (CA). ( A ) Immunohistochemical demonstration of capillary densities in chorioallantoic membranes of letrozole-treated and control mares using vWF. The capillary density tended to be greater in the control CAs than in the letrozole-treated CAs, p = 0.07; ( B ) A strong cytoplasmic signal for VEGF is noted within the chorionic epithelium of letrozole-treated group. No difference was observed in the abundance of HIF1A + cells between the groups. ( B ) Red boxes (letrozole) and blue circles (control) represent values for the capillary density scores in the chorioallantoic samples.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Fig. 4 Comparison of pro-angiogenesis properties of DPSCs and PLDSCs. (A) qRT-PCR was used to compare the expression levels of VEGF ; the expression levels were normalized to those of ACTB ( n = 3, Student's t -test). ** P < 0.01. (B) Western blotting detection of VEGF expression. (C) Immunofluorescence staining showing the protein expression of VEGF in the cytoplasm of DPSCs and PLDSCs. Scale bar = 50 mum. (D) Representative images for each group via inverted phase-contrast microscopy. Scale bar = 100 mum. (E) Analysis of tube formation parameters (number of junctions, nodes, segments and meshes; total segment length; total mesh area) using imagej ( n = 5, one-way analysis of variance). * P < 0.05 with serum-free, & P < 0.05 with DPSCs-CM. DPSCs-CM, dental pulp stem cells-conditioned medium; PLDSCs-CM, periapical lesion-derived stem cells-conditioned medium. Data are shown as the mean +- SD.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Foxp1 KO reduces vascular endothelial growth factor A (VEGF-A) expression but not pigment epithelium derived factor (PEDF). a Representative Western blot of VEGF-A in RPE-choroid lysates of Foxp1-KO (Foxp1 fl/fl Six3-Cre + ) and control mice (Foxp1 fl/fl Six3-Cre - ). alpha-tubulin was used as the loading control. VEGF-A protein was lower in Foxp1-KO mice than in the control. b - c Relative densitometry of VEGF-A and PEDF normalized to alpha-tubulin. ns: no significant difference. d - e Immunostaining of VEGF (green) and PEDF (red) in the KO and control from laser-treated mice on day 7. VEGF staining was reduced around choroidal neovascularization (CNV) in the KO mice compared to the control. Scale bar = 100 mum. RPE, retinal pigment epithelium; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; SC, sclera; *: P < 0.05, n = 4, Student's t -test

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 3 Strong cytoplasmic immunohistochemical staining of tumor cells for VEGF in a CUP case. It is shown the characteristic granular cytoplasmic staining pattern and the occasionally weak positive reaction of intervening stromal and endothelial cells (original magnification x200, counterstained with hematoxylin)

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 5. VEGF is a target gene of miR-206 in RCC cells. (A) The putative binding sites of VEGF in the miR-206 transcript. (B) The overexpression of miR-206 resulted in a significant decrease in the luciferase activity of WT-VEGF, whereas no change was observed in MUT-VEGF. (C) The expression levels of miR-206 in Caki-1 and Caki-2 cells transfected with miR-NC or miR-206 inhibitors were evaluated using reverse transcription-quantitative PCR. (D) The protein level of VEGF in transfected Caki-1 and Caki-2 cells was determined using western blot analysis. (E) The expression of VEGF was assessed in RCC and matched para-carcinoma tissues. (F) The RNA levels of miR-206 and VEGF were inversely correlated in RCC samples (r=-0.3602; P=0.0132). *P