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. 2019 Sep 6;10:2074. doi: 10.3389/fimmu.2019.02074

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Copyright © 2019 Paalme, Rump, Mädo, Teras, Truumees, Aitai, Ratas, Bourge, Chiang, Ghalali, Tordjmann, Teras, Boudinot, Kanellopoulos and Rüütel Boudinot.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Anti-hP2X4 recognizes hP2X4 in immunoprecipitation experiments, but not in western blot. (A) Left panel. Western blot using mAb19 (1), mAb27 (2), rabbit anti-rat-P2X4 polyclonal antibodies from Alomone (3) or our mouse anti-hP2X4 polyclonal antibodies (4), performed on total cell lysate of HEK293 cells transfected with hP2X4. Blots were revealed using anti-mouse and anti-rabbit HRP Abs. Right panel. hP2X4 was immuno-precipitated from the same lysate of HEK293 cells expressing human P2X4, using mAb19 (5) or mAb27 (6), and revealed by western blotting with rabbit anti-P2X4 polyclonal antibodies. Negative controls are shown in lanes 7 and 8: immunoprecipitation of non transfected HEK293 cell lysates with mAb19 (lane 7) and mAb27 (lane 8). Immunoprecipitation of P2X4 from cell lysate of HEK293 cells transfected with hP2X4 using rabbit anti-rat P2X4 polyclonal antibodies is shown in lane 9. (B) Immunoprecipitation assays using mAb27 (IgG2b; lanes 1, 4, 7) or mAb29 (IgM; lanes 2, 5, 8) or anti-IgM (lanes 3 and 6) from HEK cells overexpressing hP2X4-mcherry (lanes 1–3), mouse P2X4-mcherry (lanes 4–6), or non transfected (lanes 7, 8). P2X4 was revealed by western blotting with rabbit anti-P2X4 polyclonal antibodies. Arrows indicate hP2X4 or moP2X4 bands.