Figure 3: PfCDPK1 is a PfPKG cellular target.
(a,b) Representative spectra from the global phosphoproteomic data demonstrating that phosphorylation of serine-64 in PfCDPK1 is downregulated following Compound 2 treatment (2 μM, 60 min; Comp 2) of wild-type schizonts (a) but not in PfPKGT618Q mutant parasites (b). The inset on the left is a zoom of each spectrum to show the relative abundance of reporter ions from three experiments (126 and 127 from Expt. 1, 128 and 129 from Expt. 2, and 130 and 131 from Expt. 3). The inset on the right hand side is the fragmentation table showing the observed b-ions in orange and y-ions in blue. (c) Box plot representation of the quantitative changes in abundance of a non-phosphorylated PfCDPK1 peptide derived from wild-type and PfPKGT618Q mutant parasites and (d) of peptides spanning the serine-64 phosphorylation site in PfCDPK1. (e,f) Representative western blots and quantification of n=5 experiments ±s.e.m. showing changes in phosphorylation at serine-64 of PfCDPK1 in wild-type parasites (e) and PfPKGT618Q parasites (f) using an in-house antibody to phosphoserine-64 on CDPK1 (CDPK1-pS64). Blots were stripped and re-blotted using a structural PfCDPK1 antibody as a loading control. Student’s paired t-test was applied to test statistical significance ***P<0.001.
