Analyses of P2X1del mRNA and protein.
(A) RT-PCR of platelet (lanes 2 and 5) and Dami cell (lanes 3 and 6) RNA; the primer sets S1/AS1 and S2/AS1 are described by Greco et al.1 In lanes 1 and 4, the pcDNA3-P2X1wtplasmid was used as a PCR template. (B) Enlarged view of lanes 2 and 3. For these experiments, platelets were isolated from freshly drawn blood of at least 10 unrelated healthy volunteers. (C) Western blots of P2X1del and P2X1wt in transfected 1321N1 total cell extracts. The pcDNA3-P2X1del vector was transfected in 2 independent experiments (del1 and del2) in parallel with the pcDNA3-P2X1wt vector (wt). A nontransfected cell extract is also shown (NT). (D) Western blots of P2X1del (del) and P2X1wt (wt) proteins synthesized in a in vitro T7-coupled transcription/translation rabbit reticulocyte system. The rabbit polyclonal anti–human P2X1antibody used in these experiments was previously described.9 Bands corresponding to P2X1del and P2X1wt PCR products and proteins are indicated. Molecular weight ladder is shown on the left.
