Journal: Translational Oncology

Article Title: Noninvasive Assessment of Tumor VEGF Receptors in Response to Treatment with Pazopanib: A Molecular Imaging Study 1

doi:

Figure Lengend Snippet: Pazopanib does not block VEGFR-2-mediated uptake of scVEGF-based fluorescent tracers. (A) Pazopanib blocks scVEGF-induced VEGFR-2 tyrosine phosphorylation in PAE/KDR cells expressing 100,000 VEGFR-2 per cell. Cells were plated on 24-well plates, 75,000 cells per well, in complete culture medium, and shifted to serum-free medium 6 hours later. Top panel: After overnight serum starvation, cells were incubated for 10 minutes at 37°C with 10 nM scVEGF, either alone or in the presence of pazopanib, then lysed and analyzed by Western blot analysis with phosphotyrosine-specific antibody (pY, clone PT-66; Sigma, St. Louis, MO). Bottom panel: To ensure equal protein loading, the blot was stripped and reprobed with VEGFR-2-specific rabbit antiserum as described [22]. (B) Pazopanib does not inhibit dose-dependent uptake of scVEGF/Cy by PAE/KDR cells. A 20-minute tracer uptake by PAE/KDR (∼105 VEGFR-2/cell) and PAE (no VEGFR-2) cells in the presence or absence of 1 µM pazopanib (after 1 hour before incubation) was quantitated in triplicate wells using Starion FLA-9000 imager (FUJIFILM Medical Systems USA). (C) Pazopanib does not inhibit uptake of scVEGF/AlexaFluor594 fluorescent tracer by PAE/KDR cells. A 20-minute tracer uptake by PAE/KDR cells in the presence or absence of 10 µM pazopanib was visualized with an AxioObserver Zeiss microscope, using 43x oil objective. Control, untreated cells; Rx, pazopanib-treated cells.

Article Snippet: Reagents scVEGF and scVEGF/Cy (scVEGF site-specifically labeled with Cy5.5-maleimide) were prepared at SibTech, as described previously [ 22 ]. scVEGF/AlexaFluor594 was prepared at SibTech by site-specific conjugation of AlexaFluor594-maleimide (Invitrogen, Carlsbad, CA) to scVEGF, as described for synthesis scVEGF/Cy5.5 [ 22 ], followed by reverse phase high-performance liquid chromatography purification.

Techniques: Blocking Assay, Expressing, Incubation, Western Blot, Microscopy

Journal: Biomolecules

Article Title: Adenovirus Fibers as Ultra-Stable Vehicles for Intracellular Nanoparticle and Protein Delivery

doi: 10.3390/biom12020308

Figure Lengend Snippet: The LHB protein is attached to AlexaFluor ® 594 streptavidin Nanogold ® by the means of the biotin-streptavidin strong non-covalent bond. Cell internalization studies of the LHB protein-streptavidin gold NP conjugate in HeLa ( A ) and NIH3T3 ( B ) cells are shown. The protein construct is labeled with NHS-fluorescein (green) and further to the streptavidin-gold NP which contains an exposed Alexa595 dye (red). Cell nuclei are stained with DAPI (blue). Z-stack measurements of the cells were also obtained to ensure that the protein conjugates localize inside the cells at the same level. ( 1 ) Only the nuclei (blue) and the AlexaFluor594 (red) filters are applied in the confocal microscope. ( 2 ) Only the nuclei (blue) and the fluorescein (green) filters are active. ( 3 ) All filters are applied and the green and red signals are co-localized mergings into a yellow–orange color, indicating the effective transport of the intact complex. Scale bar: 50 μm.

Article Snippet: LHB protein molecules labeled with NHS-fluorescein (green fluorescence) were incubated with the AlexaFluor ® 594 FluoroNanogoldTM-Streptavidin gold nanoparticles (Nanoprobes), which consist of streptavidin molecules attached to the 1.4 +/10 % nm diameter Nanogold ® particle and conjugated to AlexaFluor ® 594 dye (2–3 fluorophores/streptavidin molecule)–(red fluorescence) .

Techniques: Construct, Labeling, Staining, Microscopy