Figure 5. Activation of GMRβ/JAK2 initiates at the plasma membrane. (A) A schematic diagram showing sequential immunoprecipitation of GMRβ residing on cell surface or an intracellular location. (B) HeLa cells transiently co-expressing GMRα and GMRβ were left untreated (-) or stimulated with GM-CSF for 5 or 10 min and then underwent sequential immunoprecipitation of GMRβ as outlined in (A). Immune complexes along with 1/200 input lysates were analyzed by immunoblotting using specific antibodies as indicated. (C) The extent of tyrosine-phosphorylation of GMRβ (pY-β) or of JAK2 that was associated with GMRβ (JAK2) or activated (pJAK2/JAK2) shown in panel B (lanes 4–9) plus very similar results from two other independent experiments was quantified and plotted as that described in the Materials and Methods.