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. 2009 Apr 3;106(17):7167–7172. doi: 10.1073/pnas.0811313106

Fig. 2.

Fig. 2.

Western blot analysis and PyEBL localization in P. yoelii schizont by immunostaining. (A) Western blot analysis with mAb 5B10 (lane 1), mAb 1G10 (lanes 2), and mouse serum (lane 3) specific for PyEBL against purified P. yoelii schizont extracts. A 110-kDa band was detected in both 17X and 17XL lines, with no significant difference in the protein expression level (arrowheads). This band was not detected by normal mouse serum (lane 4). Anti-AMA1 serum detected a 66-kDa band at similar levels (lane 5). (B) P. yoelii schizonts were incubated with mAb 5B10 (PyEBL), rabbit anti-AMA1 serum (AMA1), and DAPI (blue) for nuclear staining. Schizonts labeled with anti-PyEBL (5B10) were stained with FITC secondary antibody (green). Anti-AMA1 were stained with Alexa-546 secondary antibody (red). DIC images are shown in the right-hand column. The 17X line shows apical PyEBL signal colocalized with AMA1, but the region 6–substituted 17XL line shows diffused staining that does not colocalize with AMA1. (C) Immunoelectron microcopy was carried out for resin-embedded P. yoelii 17X and 17XL lines with anti-PyEBL mouse serum and secondary antibody conjugated with gold particles. PyEBL was detected in the micronemes (arrowheads) of the 17X line, but in the 17XL line it was located in the dense granules (arrows). N, nucleus; Mn, microneme; DG, dense granule; Rh, rhoptry.