Figure 2. In vitro phosphorylation of the Rh2b mutants.

(A) Targeted amino acids in the mutational analysis are the numbered serine residues and those adjacent to Ser³²³³ (HL and NY). The primary sequence of the Ser³²³³-phosphorylated synthetic peptide used for raising an antibody (anti-pRh2b) is shaded in grey. (B) In vitro radioactive [γ-³²P]ATP phosphorylation of recombinant Rh2b CPD serine mutants. Either both (Rh2b_PhosphoMut) or a single serine residue were substituted with alanine. Wild-type Rh2b incubated with uninfected RBC lysate [Rh2b_wt(RBC_control)] or with schizont lysate (Rh2b_wt) are used as a negative or positive control respectively. (C) In vitro radioactive [γ-³²P]ATP phosphorylation of additional point mutants targeting Ser³²³³ adjacent amino acids. Upper panel, autoradiography. Lower panel, Coomassie-stained SDS/PAGE. (D) Western blot analysis of recombinant wild-type and mutant Rh2b CPD that was subjected to in vitro phosphorylation in the absence or presence of ATP prior to SDS/PAGE. Western blot analysis of these proteins reveal that the anti-pRh2b antibody exclusively recognizes phosphorylated Rh2b CPD at Ser³²³³ (upper panel). Anti-GST antibody was used as a loading control (lower panel). The molecular mass is shown in kDa on the left-hand side of the blots.