Figure 5. EROS regulates P2X7 protein abundance by direct interaction and independently of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.
(A–D) P2X7 expression analysed by Western blotting of macrophages isolated from control, EROS knockout (KO) (A) and gp91phox KO mice (B), induced pluripotent stem cells (iPS)-derived macrophages control or EROS-deficient (C) and of control PLB985 cells and an EROS-deficient clone (D). (E, F) P2X7 expression in RAW264.7 cells overexpressing a FLAG-tagged EROS vector (E) and in HEK293 cells transiently expressing the specified constructs (F). (G, H) Interaction between EROS and P2X7 probed by immunoprecipitation (IP) of EROS from RAW264.7 EROS-FLAG macrophages followed by immunoblot (IB) for P2X7 (G) and by Nanoluc Binary Technology (NanoBIT) assay in live HEK293 cells expressing the LgBIT-fused EROS vector with a SmBIT-fused P2X7 vector (H). (I) P2X1 expression in macrophages isolated from EROS KO mice compared to control. n = 5 biological replicates. (J) P2X1 abundance upon co-transfection with EROS construct in HEK293 cells. Data are representative of hree independent experiments; error bars indicate SEM of triplicates. See also Figure 5—figure supplement 1 and Figure 5—source data 1–4.