|
|
Blood, Vol. 109, Issue 9, 3741-3744, May 1, 2007
Nongenomic signaling of the retinoid X receptor through binding and inhibiting Gq in human platelets
Blood Moraes et al.
109: 3741
Supplemental materials for: Moraes et al, Vol 109, Issue 9, 3741-3744
Files in this Data Supplement:
- Table S1. N-terminal sequences of human Gq peptides contain a nuclear receptor LXXLL coregulator motif (PDF, 12.8 KB) -
*Gq (AAB64301), G11 (AAB64303), G14 (AAM12617), G15 (NP_002059), G16 (AAA35860). Rat (NP_112298.1) and murine (NP_032165.2) Gq contain the identical LKLLL motifs.
- Figure S1. RXRα expression and 9cRA inhibition of platelet aggregation and Ca2+ release (JPG, 54.7 KB)
-
(A) RXR expression (brown immunogold labeling, or green immunofluorescence) in the megakaryocyte cell line Meg-01 and in human platelets. Cells were counterstained with hematoxylin. The second antibody (2nd Ab) indicates the lack of specific staining observed when specific primary antibody was omitted; RXR+pep indicates reduction in staining observed when RXR antibody was preabsorbed with RXR peptide. These data represent 3 experiments. (B-C) Examples of 9cRA (3-20 µM) inhibition of platelet aggregation (B) and Ca2+ release (C) to maximum stimulation by either ADP (B; 2-12 µM) or U46619 (C; 4 µM).
- Figure S2. 9cRA inhibition of platelet aggregation is not mediated by PPARγ (JPG, 41.4 KB)
-
(A) Typical aggregometer traces showing the decrease in light transmittance seen as platelets aggregate to (in sequence from left to right) collagen (1 µM; top panel) or U46619 (1 µM; lower panel), with the PPAR agonist 15-deoxy- 12,14-prostaglandin J2 (15d-PGJ2; 10 µM) or aspirin (ASA; 10 µM) given 3 minutes prior to collagen or U46619. (B) RXR weakly binds PPAR in a ligand-dependent fashion. Human PRP was treated with 9cRA (10 µM) for 3 minutes. Platelets lysates were immunoprecipitated (IP) with anti-PPAR and Western blotted (WB) for RXR. (C) The PPAR antagonist GW9662 (1 µM) does not affect 9cRA-inhibited platelet aggregation. 9cRA (10 µM; 3-minute pretreatment) inhibits ADP (4 µM). GW9662 has no effect on ADP-induced platelet aggregation either alone or as a 5-minute pretreatment to 9cRA. The figure represents the mean ± SEM changes in agonist ADP-induced aggregation. Data come from at least 3 separate donors. (D) Gq and RXR interact in a ligand-dependent fashion in HEK cells transfected with Gq and RXR expression plasmids. HEK 293 cells were transfected for 4 hours with expression plasmids. Cells were then split 1:2, and left for a further 20 hours. Cells were then treated with vehicle or 9cRA (10 µM, 3 minutes), before protein was extracted and subject to IP procedures. Data are representative of 3 experiments.
- Figure S3. Immunoprecipitation controls for the RXRα interaction with G proteins (JPG, 42.2 KB)
-
(A) RXR is detected (in this sample as a doublet) in human platelets by Western blot (WB: RXR) after immunoprecipitation with anti-Gq (IP: Gq), but not with control normal rabbit IgG (IP: IgG); n = 3. (B) After immunoprecipitation of platelet lysates with anti-RXR (IP: RXR), Gq is detected by Western blot (WB: Gq); however, Gi/o/t/z/gust cannot always be detected (WB: Gi/o/t/z/gust). Western blots show both Gq and Gi/o/t/zgust expression.
|
|
