Figure 3

Deglycosylation of the putative fibrinogen-related proteins in tick haemolymph and their immunodetection. 3A - Reduced D. marginatus haemolymph proteins (lane 1) were enzymatically deglycosylated (lane 2). The FReP proteins were detected using anti-DMF1 serum, which was raised against the 36 kDa protein (asterisk). After deglycosylation, additional bands appeared with sizes of 31, 33, and 34 kDa (arrows). Cross-reactivity of the serum with 79/80 kDa proteins was observed (bands with molecular weights of 58, 60, and 66 kDa - lane 1, dotted arrows). The molecular weight of these proteins shifted after deglycosylation and three bands were observed at 54, 58, and 63 kDa (lane 2, arrows). 3B - Reduced D. marginatus haemolymph proteins (lane 1) a were enzymatically deglycosylated (lane 2). The FReP proteins were detected using anti-DMF3 serum, which was raised against the 290 kDa protein. This protein is composed of two subunits which have 95 and 100 kDa (lane 1, dotted arrows). After deglycosylation (lane 2), additional bands appeared at 50 and 74 kDa (arrows). We observed cross-reactivity of the serum also in this case, when the same bands were observed for the 79/80 kDa proteins (lane 2, arrows) as in the case of anti-DMF1 serum (see Figure 2A). The protein band at 34 kDa (lane 2, asterisk) is probably a protein cleavage product. 3C - Reduced R. appendiculatus (lanes 1,2) and R. sanguineus (lanes 3,4) haemolymph proteins before (lanes 1,3) and after deglycosylattion (lanes 2,4). The FReP proteins were detected using anti-(RA)HA serum. In both tick samples (lanes 1,3), 58 kDa band was observed and a protein smear from 75 to 90 kDa (75 kDa protein and the subunits for 185 kDa protein). The deglycosylation diminished the reactivity of the protein smear and only the band at 58 kDa remained visible (lanes 2,4; arrows).