EA.hy926 cells were plated on coverslips in 12-well plate at a density of 1.4x10 5 cells per well.
Serum-starved cells were stimulated with or without 20 nM of aPC for 1 h or 10 nM a-thrombin for 1 h (Enzyme Research Laboratories, #HT 1002a), washed with cold PBS, and incubated with PBS for 10 min. Endogenous PAR1 was labeled with anti-PAR1 WEDE antibody (Beckman Coulter, #IM2584) at 1:500 for 1 h on ice, cells were treated with or without agonist, fixed for 5 min with 4% paraformaldehyde and permeabilized with 0.1% Triton-X 100. The detection of S1PR1 was determined using anti-S1PR1 antibody (Santa Cruz Biotechnology, #sc-25489) diluted at 1:100 in 0.03% BSA, 0.01% Triton-X 100 and 0.01% normal goat serum overnight at 4°C. Secondary fluorescent antibodies anti-mouse-Alexa-488 (Invitrogen, #A-11001 and anti-rabbit-Alexa-594 (Invitrogen, #A-11012) were diluted at incubated at room temperature for 1 h in 0.03% BSA, 0.01% Triton-X 100 and 0.01% normal goat serum. Slides were mounted using ProLong Gold Antifade Mountant (Invitrogen, #P10144). Confocal images were acquired using sequentially using the same settings with an Olympus IX81 spinning-disk Microscope (Tokyo, Japan) equipped with a CoolSNAP HQ2 CCD Camera (Andor) and 63x Plan Apo objective (1.4 NA) with appropriate excitation-emission filters. Line-scan analysis was performed using Image J software (NIH, Maryland, USA).
Serum-starved cells were stimulated with or without 20 nM of aPC for 1 h or 10 nM a-thrombin for 1 h (Enzyme Research Laboratories, #HT 1002a), washed with cold PBS, and incubated with PBS for 10 min. Endogenous PAR1 was labeled with anti-PAR1 WEDE antibody (Beckman Coulter, #IM2584) at 1:500 for 1 h on ice, cells were treated with or without agonist, fixed for 5 min with 4% paraformaldehyde and permeabilized with 0.1% Triton-X 100. The detection of S1PR1 was determined using anti-S1PR1 antibody (Santa Cruz Biotechnology, #sc-25489) diluted at 1:100 in 0.03% BSA, 0.01% Triton-X 100 and 0.01% normal goat serum overnight at 4°C. Secondary fluorescent antibodies anti-mouse-Alexa-488 (Invitrogen, #A-11001 and anti-rabbit-Alexa-594 (Invitrogen, #A-11012) were diluted at incubated at room temperature for 1 h in 0.03% BSA, 0.01% Triton-X 100 and 0.01% normal goat serum. Slides were mounted using ProLong Gold Antifade Mountant (Invitrogen, #P10144). Confocal images were acquired using sequentially using the same settings with an Olympus IX81 spinning-disk Microscope (Tokyo, Japan) equipped with a CoolSNAP HQ2 CCD Camera (Andor) and 63x Plan Apo objective (1.4 NA) with appropriate excitation-emission filters. Line-scan analysis was performed using Image J software (NIH, Maryland, USA).