|
|
Blood, Vol. 110, Issue 10, 3763-3772, November 15, 2007
Histone H2B as a functionally important plasminogen receptor on macrophages
Blood Das et al.
110: 3763
Supplemental materials for: Das et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 68.6 KB)
- Figure S1. Cross reactivity of anti-Plg-Rs (JPG, 42.1 KB)
-
H2B,  -enolase, annexin 2 or p11 coated plates were incubated with Fab (8 µM) of anti-peptide antibodies to the various Plg-Rs or with non-immune IgG at 22°C for 1 hr. Wells were washed and further incubated with Alexa 488 goat polyclonal anti-rabbit Fab antibody for an additional 1 hour. Wells were washed, and bound Fab was quantified in a fluorescence plate reader using an excitation wavelength of 485 nm and emission wavelength of 530 nm. Means of duplicate determinations are plotted. Each Fab binds to its target protein and has no reactivity with other Plg-Rs.
- Figure S2. Interaction between candidate Plg-Rs (JPG, 40.2 KB)
-
RAW 264.7 were pre-incubated with either Fab (8 µM) to H2B (C-terminal), -enolase, annexin 2, p11 or a combination of the anti-H2B with the Fab to each of the other Plg-Rs, for 30 minutes at 4°C followed by incubation with Alexa 488-labeled Plg (200 nM) at 4°C for 1 hour. Non-immune rabbit Fab (8 µM and 16 µM) served as a control. The cells were washed and bound Plg was measured by FACS. Specific binding was determined by subtracting the values obtained in the presence of EACA. Percent of specific binding is plotted with binding in the absence of Fab taken as 100%. Data are means ± SD of three independent experiments. The additive inhibitory effect of each Fab in combination with the H2B Fab indicates that the H2B Fab does not block Plg binding to the other candidate Plg-Rs.
|
|
