Journal: PLoS ONE

Article Title: Targeted Disruption of Core 1 β1,3-galactosyltransferase (C1galt1) Induces Apical Endocytic Trafficking in Human Corneal Keratinocytes

doi: 10.1371/journal.pone.0036628

Figure Lengend Snippet: (A) Cell surface protein was labeled with biotin at 4°C and then allowed to internalize for 15 or 25 min at 37°C. Crude postnuclear supernatants were ultracentrifuged using 5–25% Optiprep gradients and analyzed by agarose gel electrophoresis. Fraction 1 contains the lightest membranes; fraction 16 contains the densest membranes. The position of the plasma membrane and endocytic vesicles in the gradient was determined by western blot using antibodies to MUC16 and human TfR, respectively. Biotinylated protein was detected using streptavidin-peroxidase as described in . (B) Quantitative evaluation of biotin accumulation in Optiprep density gradient. The graph shows the ratio of biotinylated protein in C1galt1 shRNA and scramble shRNA cells as determined by densitometry. Band intensity in each fraction was normalized to percent total biotin loaded in each gradient. Data for each condition are reported as the mean of three independent experiments.

Article Snippet: Biotinylated protein was detected on the membranes after blocking with Tris-buffered saline Tween (TBS-T) using a biotin-streptavidin-peroxidase kit (Vectastain® ABC Kit; Vector Laboratories, Burlingame, CA).

Techniques: Labeling, Agarose Gel Electrophoresis, Western Blot, shRNA

Journal: BioMed Research International

Article Title: Isolation, Characterization, and Transplantation of Cardiac Endothelial Cells

doi: 10.1155/2013/359412

Figure Lengend Snippet: Tissue sections of the heart following cardiac endothelial cells intravenous injection. FACS sorted GFP positive EC from atrium were stained with PKH26 (red) before intravenous injection in C57BL6 mice or Rag1 null mice. Heart tissues were harvested at (A) 1 day after injection in C57BL6 mice ( n = 2), 1 month ( n = 5), and 2 months ( n = 3) in Rag1 null mice. Heart tissue sections were stained with antiCaveolin-1 followed by secondary Alexa 647 (pink) to visualize endothelial cells from 1 day post injected C57BL6 mice and 1 month post injected Rag1 null mice. In 2 month post injected Rag1 null mice, 0.5 mg/mL biotinylated-WGA was injected intravenously 30 minutes before harvesting heart tissue. Then, heart tissue sections were stained with streptavidin-Alexa 647 (magenta pseudocolor) and also stained with DAPI (blue). Bottom: although PKH-26+ GFP+ cardiac endothelial cells were detected in the heart (top), many donor cells (PKH-26+) were found in the lungs, livers, and spleens 4 weeks after transplant. Scale bar = 100 μ m.

Article Snippet: Slides were washed with Phosphate Buffer Saline (PBS, HyClone, Logan, UT), and endogenous biotin were blocked by streptavidin/biotin blocking kit (Vector, Burlingame, CA) according to the manufacturer's protocol before staining if biotinylated primary antibody is used.

Techniques: Injection, Staining