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. 2011 Oct 17;40(3):1174–1190. doi: 10.1093/nar/gkr821

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© The Author(s) 2011. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Stage-specific expression and subcellular localization of PfAlba3. (A and B) Stage-specific expression of PfAlba3 transcript was confirmed from total RNA isolated from sorbitol synchronized P. falciparum culture at ring (R), trophozoit (T) and schizont (S) stages. RT⁻ control represents reaction without reverse transcriptase. Side panel shows densitometric analysis of PfAlba3 expression at different stages of P. falciparum. Band intensities were quantified by spot densitometry using AlphaImager 3400 imaging software (Alpha Innotech Corporation) and the intensity of PfAlba3 transcript was normalized to that of seryl tRNA synthetase. (B) Western blot analysis of PfAlba3 expression in P. falciparum intra-erythrocytic stages. The sorbitol synchronized parasite lysate was resolved in 15% SDS–PAGE and subjected to western blotting using anti-PfAlba3 antibody. To confirm the equal amount of protein in parasite lysate, blot was reprobed with anti-tubulin antibody. Side panel shows densitometric analysis and intensity of PfAlba3 was normalized against tubulin. (C) Subcellular localization of PfAlba3 was confirmed by IF analysis of PfAlba3 (Red, Alexa flour 633) throughout the parasite asexual blood stages (ring, early and late trophozoite, schizont stages). (D) Nuclear localization of PfAlba3 was further confirmed by western blotting using cytoplasmic and nuclear fraction from asynchronous parasite culture. To cross check the possibilities of contamination of cytoplasm and nuclear fraction with each other, we also performed the experiment using cytoplasmic (anti-tubulin antibody) and nuclear (anti-Histone H3) marker proteins. (E) PfAlba3 also exists in the acetylated form. To confirm acetylation status of PfAlba3 in parasite, immunoprecipitated batch of protein was equally divided in two fractions and subjected to western blotting using rabbit anti-PfAlba3 antibody for first and mouse anti-acetyl lysine antibody for second fraction. Rabbit pre-immune serum was used as a control in all the experiments. Left panel showing lane 1; purified recombinant PfAlba3 (control), lane 2; immunoprecipitation with rabbit anti-PfAlba3 antibody, lane 3; immunoprecipitation with rabbit pre-immune serum. Right panel represents, Lane 4; acetylated PfAlba3 (control), lane 5; immunoprecipitation with rabbit anti-PfAlba3 antibody; lane 6; immunoprecipitation with rabbit pre-immune serum. Side panel shows the densitometric analysis of acetylated and non-acetylated PfAlba3.