|
|
Blood, Vol. 111, Issue 10, 4892-4901, May 15, 2008
CCL5-mediated T-cell chemotaxis involves the initiation of mRNA translation through mTOR/4E-BP1
Blood Murooka et al.
111: 4892
Supplemental materials for: Murooka et al
Files in this Data Supplement:
- Figure S1. CCL3/MIP1α-dependent T cell chemotaxis is not dependent on mTOR (JPG, 47.3 KB)
-
(A) Activated PB T cells were pretreated with either DMSO (carrier) or rapamycin for 1hr at the concentrations indicated, and CCL3-mediated chemotaxis measured using 10nM CCL3 (LD78 ). The data are presented as % migration, with the number of migrated cells at 10nM CCL3 taken as 100%. Representatives of two independent experiments are shown (± S.D.). * p<0.05 (B) Activated PB T cells were either left untreated or treated with 10nM CCL3 for the indicated times. Cells were harvested and lysates resolved by SDS-PAGE and immunoblotted with anti-phospho-4E-BP1 (thr-37/46) antibody. Membranes were stripped and re-probed for 4E-BP1 as a loading control. The relative fold increase of 4E-BP1 phosphorylation is shown as signal intensity over loading control to the right of each blot. Representatives of two independent experiments are shown (± S.D.)
- Figure S2. Effect of various inhibitors on T cell viability and adhesion (JPG, 67.7 KB)
-
(A) Activated T cells were treated with either DMSO (carrier), ethanol (carrier) or the specified inhibitors for 3 hrs at the concentrations indicated, stained with propidium iodide and analyzed by FACS. Cells negative for PI stain were considered viable. The data represent means ± S.D. of at least 2 independent experiments. (B) 2 × 105 T cells were either left untreated, treated with DMSO (0.1% v/v) or ethanol (0.1% v/v) for 3 hrs, plated onto fibronectin-coated wells and incubated for 2 hrs at 37°C. Cells were washed, fixed in 95% ethanol and stained with crystal violet (2% w/v). 100µl of solubilization buffer was added and the absorbance read at 570nm. Data are representative of two independent experiments performed in triplicate. The data show that the presence of DMSO or ethanol as a carrier did not affect T cell adhesion to fibronectin.
|
|
