Proteinase-K Protection Assay for P2Y1 Receptor

The proteinase-K protection assay was performed as described [25 (link), 26 (link)]. HeLa cells were plated in six-well culture dishes (4.0 X 10⁵ cells/well), grown overnight at 37°C and transfected with FLAG-P2Y₁ wildtype (WT) or FLAG-P2Y₁ Y155A, as described above. Cells were incubated with or without 10 μM ADP and 1 mM Leupeptin (Calbiochem). Cells were placed on ice and incubated for 5 min with PBS, harvested and gently permeabilized using 6.5 μg/ml digitonin (Thermo Fisher Scientific). Membranes were collected by centrifugation and resuspended in buffer (100 mM K₂HPO₄/KH₂PO₄, 5 mM MgCl₂, 250 mM sucrose). Membranes were then divided into three aliquots either left untreated, treated with 2.5 ng/ml proteinase-K, or treated with proteinase-K supplemented with 0.1% Triton-X 100 for 10 min at room temperature. After treatments, samples are diluted with 100 μl 2X SDS sample buffer containing 20 mM PMSF and analyzed by immunoblotting as described above.