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Blood, Vol. 114, Issue 9, 1974-1986, August 27, 2009
Endothelial cell protein C receptor cellular localization and trafficking: potential functional implications
Blood Nayak et al.
114: 1974
Supplemental materials for: Nayak et al
Active site-inactivated FVIIa and active site-inactivated APC
Active site-inactivated FVIIa and active site-inactivated APC were prepared by incubating FVIIa and APC, respectively, with 100 fold molar excess of D-Phe-L-Phe-L-Arg chloromethyl ketone (Calbiochem, San Diego, CA) for 1 h at room temperature and then removing the unreacted inhibitor by extensive dialysis. The inactivated FVIIa and APC had no detectable protease activity as measured in amidolytic activity assay. Construction of EPCR-GFP
Human EPCR cDNA cloned in pZeoSV (pZeoSC-EPCR219)16 was used as template to generate full-length EPCR cDNA and EPCR cDNA lacking 45 bp (signal peptide sequence) at its N-terminus by PCR amplification. The forward and reverse primers used for full length EPCR gene amplification were 5′ CGC CTC GAG AGG ATG TTG ACA ACA TTG CTG CCG ATA CTG C 3′ and 5′ CGG TGG ATC CCG ACA TCG CCG TCC ACC TGT GCA CAG GAA G 3′, respectively. The forward and reverse primers used for the amplification of N-terminal 45 bp truncated EPCR gene were 5′ CGC CTC GAG ACC ATG TTT TGT AGC CAA GAC GCC TCA GAT G 3′ and 5′ CGG TGG ATC CCG ACA TCG CCG TCC ACC TGT GCA CAG GAA G 3′, respectively. Nucleotides shown in bold are the restriction endonuclease site for restriction endonuclease Xho1 and BamH1 in the forward primer and reverse primers, respectively. The amplified PCR products were digested with Xho1 and BamH1 restriction enzymes and ligated with pEGFPC1 vector predigested with Xho1 and BamH1 restriction enzymes to construct EPCR-GFP fusion plasmid. Expression of wild-type and mutant rab11 in CHO-EPCR cells
To study the effect of wild-type rab11 and rab11 mutants on the trafficking of FVIIa and APC, CHO-EPCR cells were transiently transfected with wild-type, constitutively active (rab11 Q70L) and dominant negative (rab11 S25N) form of rab11 cloned in pCDNA3 mammalian expression vector controlled under CMV promoter. The cells were transfected using FuGENE HD transfection reagent according to the manufacturer’s protocol and used in experiments 48 h after the transfection.
Files in this Data Supplement:
- Figure S1. EPCR expression in endothelial cells is independent of confluency status (JPG, 422 KB)
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Monolayers of HUVEC at various stages of confluency were fixed and left intact (non-permeabilized) or permeabilized with 0.1% Triton X-100 for 10 min. Both non-permeabilized and permeabilized cells were immunostained with EPCR mAb (JRK1500, 10 µg/ml), followed by Rhodamine Red-conjugated anti-mouse IgG. Immunofluorescence was analyzed by confocal microscopy. A single panel shown in the last row is a digital magnification of the selected area from the image immediately above to it to show punctuate distribution of EPCR at the cell surface.
- Figure S2. Intracellular localization of EPCR in CHO-EPCR cells (JPG, 549 KB)
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Permeabilized CHO-EPCR cells were immunostained with EPCR mAb and an organelle-specific antibody. Left panel represents organelle specific staining, middle panel represents EPCR specific staining, and the right panel shows the overlay image (co-localization) of organelle-specific marker and EPCR. Organelle-specific antibodies used were as follow: anti-EEA1 and anti-rab5 for early endosomes, anti-LAMP1 for lysosomes, Giantin for the Golgi and rab11 for the recycling compartment.
- Figure S3. Internalization of APC, FVII and protein C in endothelial cells (JPG, 715 KB)
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HUVEC were incubated were with 50 nM of AF488-APC (A), AF488-FVII (B) or AF488-protein C for varying time intervals at 37°C. The cells were washed with calcium-containing buffer and then fixed. The permeabilized cells were immunostained with non-blocking EPCR mAb. Left panel represents EPCR staining, middle panel shows the fluorescence of AF488-conjugated ligands, and right panel depicts the overlay image of left and middle panels plus nuclear staining with DAPI. Arrows show the accumulation of AF488-FVIIa or AF488-APC with EPCR in the recycling endosomal compartment.
- Figure S4. Internalization of APC bound to EPCR in CHO-EPCR cells (JPG, 522 KB)
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CHO-EPCR cells were exposed to AF488-protein C (50 nM) for 1 h at 4°C. At the end of 1 h, the supernatant was removed; the cells were washed quickly with calcium-containing buffer to remove the unbound ligands and then transferred to 37°C to allow internalization of the surface bound ligand. Varying times at 37°C, the cells were fixed, permeabilized and immunostained with non-blocking EPCR mAb. Left panel represents AF488-APC, middle panel shows EPCR staining and right panel depicts the merged image of AF488-APC and EPCR.
- Figure S5. Internalization of active site-inhibited FVIIa and APC in CHO-EPCR cells (JPG, 745 KB)
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CHO-EPCR cells were incubated with AF488 conjugated active site-inhibited FVIIa (AF488-FVIIa/ASI) (50 nM) (A) or AF488-conjugated active-site inhibited APC (AF488-APC/ASI) (50 nM) (B) for varying time intervals at 37°C. The cells were fixed, permeabilized and immunostained with non-blocking EPCR mAb. Left panel represents AF488-FVIIa/ASI fluorescence, middle panel shows EPCR staining and right panel depicts the overlay image of AF488-FVIIa/ASI and EPCR staining.
- Figure S6. Internalized FVIIa sorts to the recycling compartment (JPG, 258 KB)
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CHO-EPCR cells were exposed simultaneously to AF488-FVIIa (50 nM) and AF555-transferrin (300 nM) for 5 to 30 min. The cells were fixed, permeabilized and analyzed by confocal microscopy. Insets in the merged image panel show the magnified view of colocalization of AF488-FVIIa and AF555-transferrin beneath the plasma membrane at 5 min and in the recycling compartment at 30 min.
- Figure S7. Specific binding of human FVIIa to mouse EPCR (JPG, 66.1 KB)
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Wild-type 293 cells or 293 cells stably transfected with mouse EPCR cultured in 12-well plate were incubated with 10 nM of 125I-labeled human FVIIa for 2 h at 4°C. At the end of 2 h, the supernatant was removed, the cells were washed 4 times with calcium containing buffer and the cell associated radioactivity was eluted with low pH buffer (0.1 M glycine, pH 2.3). mEPCR expressing cells were pre-incubated with control vehicle, mEPCR blocking or non-blocking mAb (10 µg/ml) for 30 min at room temperature before 125I-FVIIa was added to the cells.
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