Fig. 2.

Establishment of P2X7-, Xk-, or Vps13a-deficient cell lines. (A) Western blot analysis. The P2rx7 gene in Tmem16f^−/− WR19L cells was knocked out to generate 16f^−/−P2rx7^−/− cells (DKO). DKO was transformed with mouse P2X7 to establish DKO-P2X7. The Xk gene in DKO-P2X7 was knocked out in Xk^−/−, and Xk^−/−DKO-P2X7 was transformed with FLAG-tagged mouse Xk to generate Xk^−/−DKO–P2X7–Xk (Xk^−/−-Xk). In Vps13a^−/−, the Vps13a gene was knocked out in DKO-P2X7, and Vps13a^−/−DKO-P2X7 was transformed with mouse Vps13a in Vps13a^−/−–Vps13a. In Xk^−/−Vps13a^−/−, Xk, and Vps13a genes were knocked out in DKO-P2X7. Approximately 4.7 μg of protein per lane (except for Xk^−/−–Xk containing 94 ng of protein) of whole-cell lysates was separated by SDS-PAGE. Western blotting was performed with Abs against P2X7, Xk, and Vps13a, and membranes were stained with CBB. The red arrowhead shows Xk. *, non-specific band. (B) The cell-surface expression of P2X7. DKO, DKO-P2X7, Xk^−/−, Xk^−/−–Xk, Vps13a^−/−, Vps13a^−/−–Vps13a, and Xk^−/−Vps13a^−/− cells were stained with Alexa 647–anti-P2X7 Ab. The Alexa 647-staining profiles in the SYTOX Blue-negative population are shown.