RhAG binds Rh at the plasma membrane in basophilic erythroblasts. (A) 1 × 10⁶ cells from the indicated time points in differentiation were either treated (internal) or not (total) with 500 μg/mL pronase before lysis (T = time in hours). Proteins were immunoblotted with LA1818. Uncleaved pronase-resistant intracellular RhAG is detectable at the early stages of erythropoiesis. (B) 1.5 × 10⁷ cells at the indicated time points in differentiation were used for total (T), internal (I), or surface (S) immunoprecipitations, respectively, using the RhAG antibody LA1818 as described in “Immunoprecipitations and total cell lysates.” Proteins were detected by immunoblotting with polyclonal antibodies raised against the C-termini of Rh and RhAG. (C) Lysate from 3 × 10⁷ erythroblasts differentiated for 48 hours and treated or not with 500 μg/mL pronase were used for RhAG (LA1818) immunoprecipitations. Rh is coimmunoprecipitated with RhAG from total cell lysates but not from pronase-treated cell lysates (internal pool immunoprecipitate). (D) Coimmunoprecipitation of RhD (lane 4) and RhCe (lane 6) with RhAG using LA1818 (RhAG, surface immunoprecipitate [IP]; see “Immunoprecipitations and total cell lysates”) but not with the IgG1 control immunoprecipitate (lane 5) from the plasma membrane of HEK293T cells transfected with the indicated combinations of expression constructs encoding human RhD, RhCe, and RhAG. Lanes 1-3 represent

of the total cell lysates used in the immunoprecipitations.