Fig. 1. Characterization of TbCentrin4. (A) Characterization of antibodies against TbCentrin4. Total lysates containing equal amounts of protein (12 µg) from control procyclic cells (lanes labeled 1) or cells stably expressing TbCentrin4-YFP (lanes labeled 2) were fractionated, separated by electrophoresis and immunoblotted with the indicated antibodies. Note that antibody against GFP (which also recognizes YFP; second panel) recognized the same fusion protein as polyclonal antibodies against TbCentrin4 (third panel), which also recognized two proteins at 14 and 16 kDa (arrows, fourth panel, longer exposure of third panel). These represent endogenous TbCentrin4 and were not recognized by the monoclonal antibody 20H5 (which recognizes co-migrating TbCentrin1 and TbCentrin2; first panel). (B) Maximum-likelihood sequence tree of centrins and calmodulins. Numbers on branches represent bootstrap support values. Branches supported by less than 50% of the bootstrap replicates were collapsed to form a multifurcation. L_major, Leishmania major; L_donovani, Leishmania donovani; CALM, calmodulin; Cen, centrin. We used the centrin nomenclature suggested by Selvapandiyan et al. (Selvapandiyan et al., 2007) with the exception of the TbCentrins (He et al., 2005), which are suffixed `_He' to highlight our results. The numbers on the right indicate the number of predicted EF-hand domains in those proteins grouped by the numbered square brackets.