The cytoplasmic domain of STEVOR is phosphorylated by PKA. (A) Schematic representation of STEVOR proteins and alignment of flanking sequences of the 3 NetPhos-predicted serine phosphorylation sites for all members of the STEVOR family. Serine S₁₂₃, S₁₆₅, and S₃₂₄ (in red) are numbered regarding the consensus sequence. (B) In vitro PKA phosphorylation assay for 3 peptides corresponding to a typical PKA phosphorylation substrate (Kemptide) and to the predicted-phosphorylation site on the C-terminal domain of STEVOR (Stevor S₃₂₄). As a negative control, an alanine residue was substituted to the serine 324 (Stevor A₃₂₄). The 3 peptides were incubated with a recombinant bovine PKA in the presence of 32[P]ATP, with or without PKA inhibitors H89 or KT5720. Phosphorylation signal (mean ± standard deviation [SD]) under different conditions is reported relative to kemptide phosphorylation from 3 independent experiments. a.u., arbitrary units. (C) In vitro PKA phosphorylation assay for the the Stevor S₃₂₄ and the Stevor A₃₂₄ peptides incubated with 40-hpi schizont extracts (control line) in the presence of 32[P]ATP, with or without H89. Phosphorylation signal (mean ± SD) under different conditions is reported relative to Stevor S₃₂₄ peptide phosphorylation from 3 independent experiments. (D) Immunoprecipitation of 40-hpi schizont lysates from the Full-myc line with anti-myc, anti-GST, and anti-phospho-PKA antibodies. Immunoblot was probed with a rat antibody directed against STEVOR (PFC0025c). (E) Immunoprecipitation of stage III GIE lysates from the Full-Ty1 line with anti-GST and anti-phospho-PKA antibodies. Immunoblot was probed with a rabbit antibody directed against STEVOR (PFC0025c). (F) Immunoprecipitation of 40-hpi schizont lysates from the Full-myc line with anti-GST and anti-myc antibodies. Immunoblot was probed with a rabbit anti-phospho-PKA antibody.