Translation of <i>L. infantum</i> developmentally regulated transcripts under temperature and acidic pH stresses.

<p>(A) Northern blot hybridization of <i>L. infantum</i> RNA isolated from sucrose gradient fractions (F, M, L and H are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035085#pone-0035085-g005" target="_blank">Figure 5</a>) to assess the association of the amastigote-specific <i>A2</i> transcript with the ribosomes under temperature stress or acidic pH stress or a combination of both stresses. The ethidium bromide-stained gels used for northern blot analysis served as a loading control (rRNA is indicated). (B) Western blot analysis of <i>L. infantum</i> protein lysates using a monoclonal anti-A2 antibody to determine expression levels of A2 protein under different stresses, including high temperature, low pH and Tg-induced ER stresses. Controls using <i>L. infantum</i> promastigotes (Pro) (non stress) or axenic amastigotes (Ama) were included for both northern and western blots. (C) Northern blot hybridization of <i>L. infantum</i> total RNA to determine <i>A2</i> mRNA expression during amastigote to promastigote differentiation for up to 48 hrs. (D) Northern blot hybridization of <i>L. infantum</i> RNA isolated from sucrose gradient fractions to determine the association of the <i>A2</i> mRNA with monosomes or translating polysomes. (E) Western blot with an anti-A2 antibody of <i>L. infantum</i> amastigotes transferred into MAA-20 medium at 25°C and pH 7.3 for up to 5 hours to allow its differentiation into promastigote forms. The anti-alpha-tubulin antibody was used as loading control. Data shown here are representative of three independent experiments with similar results.