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LamAT-GFP localizes to the Leishmania nucleus.

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posted on 23.06.2011, 00:48 by Ben L. Kelly, Gyanendra Singh, Ashok Aiyar

(A) Schematic of the LamAT-GFP chimera stably expressed in L. amazonensis. (B) Immunoblot analysis of lysates from LamAT-GFP transfectants and wildtype controls using anti-GFP antisera ab290. Blots were stripped and probed with anti-LACK antisera as a loading control (C) Parasites were fixed, cytocentrifuged onto slides, immunostained with anti-GFP (ab290), followed by a Texas Red-conjugated secondary antibody and counterstained with DAPI. Parasites were then visualized at 630X under DIC illumination or by indirect immunofluorescence. Bars indicate a scale of 10 µm. (D) Immunoblotting was performed on nuclear and cytoplasmic extracts of control and LamAT-GFP expressing L. amazonensis and probed with antisera against GFP and histone H3, as indicated. (E) Visualization of axenic amastigote forms of L. amazonensis expressing LamAT-GFP. LamAT-GFP was visualized by direct fluorescence, and nuclei were counterstained with DAPI. Bars indicate a scale of 10 µm.

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