Fig. 4. In vivo Myristoylation assay of truncated Tail fragment of BFSP1:

A: Truncated Tail fragment of BFSP1 was co-expressed with CaNMT in E. coli in the presence of azido-myristate. CLICK-chemistry was used to detect myristolyated proteins and detected in the gel after fluorescently labelled with Cy3-TAMRA. PfARF1 (Plasmodium falciparum ADP-ribosylation factor-1) was included as a positive control of myristoylation. Track1 and 2, Pre- and post-induction of PfARF expression in E.coli; Tracks 3 and 4, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli; Tracks 5 and 6: Pre- and post-induction of G434-P548 BFSP1 expression in E.coli; Tracks 7 and 8, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli; Tracks 9 and 10, Pre- and post-induction of PfARF with C.albicans NMT expression in E.coli fed with azido-myristate followed by CLICK chemistry detection; Tracks 11 and 12, Pre- and post-induction of BFSP1 G434-P548 with C.albicans NMT expression in E.coli fed with azidomyristate followed by CLICK chemistry detection. Notice the positive bands in tracks 10 and 12 only.

B. Km and Vmax determination for recombinant human NMT1 and 2 for the BFSP1 sequence G434- F441.

C. Characterization of the polyclonal antibodies NP-Tail and NP53 using human lens samples. The soluble protein fraction (S1), the plasma membrane cytoskeleton complex (PMCC) and plasma membrane fraction (P3) were isolated from the human cortex and separated by SDS PAGE and the 10% (w/v) polyacrylamide gel prior to either staining with Coomassie Blue or immunoblotting and probing with the polyclonal antibodies 3241, NP-53 and NP-Tail. Notice that the major immunoreactive band detected in the P3 sample by the NP-tail antibodies would correspond to a C-terminal fragment of less than 25 kDa. Marker proteins (m) are indicated (•) and correspond to 250, 130, 100, 70, 55, 35, 25 and 15 kDa as per PageRuler prestained standards.