rabbit anti pan vegf a  (Santa Cruz Biotechnology)

Journal: PLoS ONE

Article Title: Detection of VEGF-A xxx b Isoforms in Human Tissues

doi: 10.1371/journal.pone.0068399

Figure Lengend Snippet: A. Plasmids containing VEGF-A 165 b or VEGF-A 165 a sequence were amplified using primers in exon 8b and exon 7. Two different sized products were generated. B. Densitometric analysis of published RT-PCR gels using plasmids containing VEGF-A 165 b and VEGF-A 165 a and primers in exon 7 and exon 8b. 13/15 show higher intensity for VEGF-A 165 a. p<0.001 paired t test. C. Example of failure of amplification of the VEGF-A 165 b isoform. Two parallel PCR reactions were run on cDNA and plasmid DNA. On the first (at 4mM MgCl 2 ) no VEGF-A 165 b was generated in the cDNA, or when both VEGF-A 165 a and VEGF-A 165 b plasmids were used as positive controls. In the second (at 5mM MgCl 2 ) VEGF-A 165 b was generated from cDNA from PC3 cells where SRPK1 was knocked down, and when both templates were included. D. Example of failure of amplification of the VEGF-A 165 b isoform in one PCR machine (and failure of template), but not in a second machine. E. Example of failure of amplification of VEGF-A 165 b product after freeze-thawing of cDNA derived from fresh normal human lung fibroblasts. Chromatogram confirms VEGF-A 165 b sequence from lower band from top gel from patient sample in lane 1 (chromatograms from the other samples also confirmed VEGF-A 165 b sequence).

Article Snippet: The immunoprecipitate (IP) and 50 μg of the crude extract were subjected to SDS-PAGE and immunoblotting using a rabbit polyclonal anti-pan-VEGF antibody (A20, Santa Cruz).

Techniques: Sequencing, Amplification, Generated, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Derivative Assay

Journal: PLoS ONE

Article Title: Detection of VEGF-A xxx b Isoforms in Human Tissues

doi: 10.1371/journal.pone.0068399

Figure Lengend Snippet: A. Sequence of the VEGF 3′ exon sequence. (i) Exon 7 (red) contains the same last three nucleotides (underlined) as the last three nucleotides of exon 8a (blue, underlined), requiring specific PCR primers that extend into exon 7 (arrow). (ii) mispriming (VEGF-A 165 a -specific primers priming on VEGF-A 165 b, and VEGF-A 165 b -specific primers priming on VEGF-A 165 a) can occur both ways round if the conditions are not tested. B. Published control PCR gels demonstrating specificity of primer conditions. The original description of VEGF-A 165 b describing conditions at which VEGF-A 165 b is not misprimed in the presence of 100ng VEGF-A 165 a (lane highlighted by arrow), but still able to amplify 0.1ng VEGF-A 165 b. C. Annealing temperature dependence of the specificity of the isoform specific primers. Only at >62°C is specificity resolved. D. qPCR using VEGF-A 165 a specific primers on VEGF-A 165 a and VEGF-A 165 b plasmid E. qPCR using VEGF-A 165 b specific primers on VEGF-A 165 a and VEGF-A 165 b plasmid.

Article Snippet: The immunoprecipitate (IP) and 50 μg of the crude extract were subjected to SDS-PAGE and immunoblotting using a rabbit polyclonal anti-pan-VEGF antibody (A20, Santa Cruz).

Techniques: Sequencing, Control, Plasmid Preparation

Journal: PLoS ONE

Article Title: Detection of VEGF-A xxx b Isoforms in Human Tissues

doi: 10.1371/journal.pone.0068399

Figure Lengend Snippet: A. C t max-C t for cDNA extracted from prostate cancer (PC3) cells with lentiviral knockdown of SRPK1 or scrambled. B. Amount of VEGF calculated from standard curves in . C. Amount of VEGF-A 165 b identified by Exon 8b primers (VEGF-A 165 b) or that calculated from mispriming of VEGF-A 165 a. D. Proportion of VEGF that is VEGF-A 165 a or VEGF-A 165 b in control and knockdown cells. Values are Mean±SEM (n = 2). 3E. qPCR for VEGF-A 165 a on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar). 3F qPCR for VEGF-A 165 b on commercially available cDNAs from 2 different companies (open bars) or cDNA reverse transcribed from freshly extracted human kidney RNA (solid bar).

Article Snippet: The immunoprecipitate (IP) and 50 μg of the crude extract were subjected to SDS-PAGE and immunoblotting using a rabbit polyclonal anti-pan-VEGF antibody (A20, Santa Cruz).

Techniques: Knockdown, Control, Reverse Transcription

Journal: PLoS ONE

Article Title: Detection of VEGF-A xxx b Isoforms in Human Tissues

doi: 10.1371/journal.pone.0068399

Figure Lengend Snippet: A. Western blot using LiCor Odyssey to simultaneously image pan-VEGF and VEGF-A 165 b probed western blot. Two different podocyte samples, and a primary RPE sample were run on a gel and probed with antibodies to VEGF-A 165 b (mouse monoclonal anti-CTRSLTRKD, and 680nm-donkey anti-mouse, top image) and pan-VEGF (rabbit polyclonal anti-VEGF, and 800nm-donkey anti-rabbit, middle image). The bottom image is the pseudocoloured combined image (600nm green, 800nm red). Note the red VEGF 165 , but yellow VEGF-A 165 b. MWM = molecular weight marker. d = dimer, m = monomer. B. Protein extracted from human cell lines (adenoma and adenocarcinoma(AC)) subjected to immunoprecipitation (IP) for VEGF-A 165 b and immunoblotting (IB) for total VEGF-A. A clear strong band was seen in the IP for both cell types at ∼23kDa and ∼46kDa, consistent with the IP for recombinant human VEGF-A 165 b. A weaker band was seen in the input protein (not subjected to IP), and a second band slightly higher in the AC. A weak band at approximately 56kDa and 28kDa was seen in all lanes subjected to IP, including the VEGF-A 165 a band, but not seen in the recombinant human VEGF-A 165 b not subjected to IP, indicating that this is cross reactivity with the IgG. This band was clearly above the VEGF-A 165 b bands. C. Protein extracted from human cell lines (adenoma and adenocarcinoma(AC)) subjected to immunoprecipitation (IP) for VEGF-A and immunoblotting (IB) for VEGF-A 165 b. A clear strong band was seen in the IP for both cell types at ∼23kDa, the same size as recombinant human VEGF-A 165 b. In the input a band at ∼46Da was seen predominantly, for both cell types, labelled as VEGF-A 165 b dimers. D. Mouse tissues probed with VEGF-A 165 b antibody detect mouse IgG due to the secondary antibody. Top image, western blot of mouse tissues, recombinant mouse IgG or human VEGF-A 165 b or VEGF-A 165 b probed with mouse anti-CTRSLTRKD, and 680nm-donkey anti-mouse IgG. Bottom image blot of same tissues, probed without primary antibody. The same bands are seen in the mouse tissues. Spl = spleen, Col = colon, Hrt = heart, Lng = lung, Liv = liver, Kid = kidney.

Article Snippet: The immunoprecipitate (IP) and 50 μg of the crude extract were subjected to SDS-PAGE and immunoblotting using a rabbit polyclonal anti-pan-VEGF antibody (A20, Santa Cruz).

Techniques: Western Blot, Molecular Weight, Marker, Immunoprecipitation, Recombinant

Journal:

Article Title: Maintaining retinal astrocytes normalizes revascularization and prevents vascular pathology associated with oxygen induced retinopathy

doi: 10.1002/glia.20900

Figure Lengend Snippet: Injection of small quantities of bFGF or VEGF can protect retinal astrocytes and rescue the retinal vasculature in the OIR model. A) bFGF and VEGF is secreted by astrocytes and is present in the astrocyte-conditioned, serum-free media. B-C) Injection of bFGF (B) or VEGF (C) can rescue the retinal vasculature in the OIR model in a dose dependent manner (N ≥ 10 for each condition). D) The observed vascular rescue effects are preceded by protection of the endogenous astrocytes from hypoxia-induced degeneration within the vascular obliterated zones at P14 E) Quantification of the observed astrocytic rescue effects (N ≥ 10 for each condition; for all observers p values ≤ 0.001 for bFGF or VEGF compared to vehicle injection). F) Filopodia are extended from the endothelial cells planar to the protected astrocytes within the vaso-obliterated retina. These extensions can mediate potential guidance along the astrocytic template and suggest an explanation for the observed normalized revascularization associated with astrocyte protection in the vaso-obliterated zone. Each slightly opaque square indicates the area shown in the adjacent, higher magnification image. (Size bars = 100 μm)

Article Snippet: After blocking with 5% milk buffer in PBS + 0.1% Tween, the blots were incubated with rabbit anti-mouse bFGF (NOVUS, 008-100) or rabbit anti-mouse pan VEGF (Chemicon, AB1876).

Techniques: Injection