α-Actinin TvACTN3 of Trichomonas vaginalis Is an RNA-Binding Protein That Could Participate in Its Posttranscriptional Iron Regulatory Mechanism
Figure 2
Identification of T. vaginalis TvACTN3 by 1-D gel electrophoresis and mass spectrometry. (a) UV Cross-linking assay of 32P-labeled IRE-fer and IRE-tvcp4 transcripts and T. vaginalis cytoplasmic extracts grown under iron-depleted conditions (Tv-L) (lanes 2 and 7, resp.). The specificity of the interaction was demonstrated by treatment with proteinase K (10 μg, Promega) (lanes 3 and 8) and RNases (lanes 4 and 9) and a complete binding reaction using unlabeled IRE-fer and IRE-tvcp4 (lanes 5 and 10) or only the labeled probes (lanes 1 and 6). Molecular mass markers are indicated in kilodaltons (kDa). Arrowheads indicate the positions of the RNA-protein complex bands of 135, 110, 70, and 45 kDa. A representative result of three independent experiments with similar results is shown. (b) Cytoplasmic extracts of T. vaginalis grown in iron-depleted medium were Coomassie blue-stained (lane 1) or transferred onto NC membranes for Western blot (WB) assays (lanes 2, 3, and 4); and incubated with preimmune (PI) serum (negative control, lane 2), anti-chicken α-actinin (α-chACTN, lane 3), or anti-rat-IRP-1 (α-IRP1, lane 4) antibodies. (c) Representative supershift assays. Radiolabeled IRE-fer and IRE-tvcp4 RNA probes (lanes 1 and 7, resp.) were incubated with cytoplasmic proteins from T. vaginalis grown under iron-depleted conditions without (lanes 2 and 8) or with the α-chACTN polyclonal antibody (1 μg) (lanes 3 and 9), (2 μg) (lanes 4 and 10), or with the anti-bACTN monoclonal antibody (1 μg) (lanes 5 and 11), (2 μg) (lanes 6 and 12), or with a nonrelated antibody (α-TvTIM2r; lane 13). The arrowhead indicates RNA-protein complex. A representative result of three independent experiments yielding similar results is shown.
