VEGF expression determined by Western blot and immunoprecipitation.

<p>A. Western blot using LiCor Odyssey to simultaneously image pan-VEGF and VEGF-A₁₆₅b probed western blot. Two different podocyte samples, and a primary RPE sample were run on a gel and probed with antibodies to VEGF-A₁₆₅b (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 VEGF₁₆₅, but yellow VEGF-A₁₆₅b. 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-A₁₆₅b 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-A₁₆₅b. 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-A₁₆₅a band, but not seen in the recombinant human VEGF-A₁₆₅b not subjected to IP, indicating that this is cross reactivity with the IgG. This band was clearly above the VEGF-A₁₆₅b bands. C. Protein extracted from human cell lines (adenoma and adenocarcinoma(AC)) subjected to immunoprecipitation (IP) for VEGF-A and immunoblotting (IB) for VEGF-A₁₆₅b. A clear strong band was seen in the IP for both cell types at ∼23kDa, the same size as recombinant human VEGF-A₁₆₅b. In the input a band at ∼46Da was seen predominantly, for both cell types, labelled as VEGF-A₁₆₅b dimers. D. Mouse tissues probed with VEGF-A₁₆₅b antibody detect mouse IgG due to the secondary antibody. Top image, western blot of mouse tissues, recombinant mouse IgG or human VEGF-A₁₆₅b or VEGF-A₁₆₅b 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.