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. 2014 Jul;86(1):1–11. doi: 10.1124/mol.114.091595

Fig. 4.

Fig. 4.

PAR4 coimmunoprecipitates with P2Y12 upon PAR4-AP stimulation, but PAR4SFT does not in HEK293T cells. (A) HEK293T cells stably expressing FLAG-tagged P2Y12 were transiently transfected with V5-PAR4-GFP or HA-PAR4SFT-GFP. Forty-eight hours post-transfection, cells were left untreated or stimulated with AYPGKF (200 μM) 10 minutes at 37°C with and without MeSAMP (150 μM) 10 minutes at room temperature. Cell were lysed, immunoprecipitated with anti-FLAG antibody (Abcam; 1:1000) and immunoblotted (IB) for antibody to GFP (MBL; 1:500). (B and C) Surface expression of P2Y12, PAR4, and PAR4SFT in HEK293T cells. HEK293T cells stably expressing FLAG-P2Y12 (B) and transiently expressing HA-PAR4 or HA-PAR4SF (C) were harvested in Hanks' balanced salt solution + 0.02% EDTA, washed with 1× PBS, and resuspended to be fixed in DMEM + 5% fetal bovine serum, stained with anti-FLAG and anti-HA, and probed with Alexa 488 secondary antibody to test the membrane expression of these receptors; cells were fixed poststaining. (D) HEK293T cells transiently expressing V5-PAR4-GFP and FLAG-P2Y12 were treated with apyrase (2 U/ml) and AYPGKF (200 μM), lysed, and immunoprecipated with anti-FLAG (2 μg/ml) and immunoblotted with anti V5 (1:1000).