Figure 1

Functional EPOR validation and EPOR/EpoR detection using ctEPOR-AB in Western blots. (A) Incubation of EPO-dependent UT-7 cells (after 12 h of EPO deprivation) for 15 min with increasing EPO concentrations inducing STAT5 phosphorylation. (B) Incubation of OCIM-1 cells for 15 min with increasing EPO concentrations inducing STAT5 phosphorylation. (C) Cell counts of EPO-dependent UT-7 cultures 72 h after seeding in the presence and absence of EPO (n = 6, mean ± SEM; p < 0.0001). (D) Cell death in EPO-dependent UT-7 cultures 72 h after seeding in presence and absence of EPO (n = 5, mean ± SEM; p < 0.03). (E) Incubation of EOC-20 cells transduced with HA-tagged human EPOR for 10 min with increasing EPO concentrations inducing MAPK phosphorylation. (F) EPOR Western blot using ctEPOR-AB on transfected HEK293 FT cell lysates (truncated HA-EPOR: human EPOR lacking the C-terminus, HA-tag at the N-terminus; control vector: anchored human EPOR without N-terminus and C-terminus). (G) HA Western blot of the same transfected HEK293 FT cell lysates used in (F). (H) EPOR Western blot using ctEPOR-AB on EOC-20 cell lysates transduced with N-terminally HA-tagged human EPOR and respective controls. (I) HA Western blot of the same EOC-20 cell lysates used in (H). (J) EPOR Western blot using ctEPOR-AB on UT-7 and OCIM-1 cell lysates. (K) Flow cytometry of fixed UT-7 cells stained with ctEPOR-AB and Alexa Fluor 488 donkey anti-rabbit secondary AB; as control secondary AB only. (L) EPOR detection in human placenta and human fetal brain using ctEPOR-AB. (M) Detection of murine EpoR in transfected HEK293 FT cells overexpressing murine EpoR, mouse fetal liver and mouse primary oligodendrocytes using ctEPOR-AB. (N) Lentivirus-mediated conditional EpoR knockout in primary EpoR-fl/fl mouse astrocytes; anti-α tubulin as stably expressed comparator.