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Fig. 2. pixie encodes an ABC protein required for growth and translation. (A) A tree diagram illustrating the sequence similarity of the ABC domains of Pixie with those of other ABC-E and ABC-F proteins from Drosophila (Dm), Saccharomyces cerevisiae (Sc) and humans (Hs). (B) Predicted domain structure of Pixie, showing approximate positions of mis-sense mutations used in this study: pixL24 (Pro38Leu), pixL17 (Ala77Thr), pixL35 (Pro146Leu), pix3c2 (Gln231Leu) and pix3c3 (Gly316Asp). Pro38 (L24) and Ala77 (L17) are highly conserved consensus residues of the iron-sulphur binding domains. (C) Graphs show the impact of various dsRNAi treatments on global translation, measured as [35S]cysteine and methionine incorporation into total cellular protein, and represent combined data from three independent experiments. Relative translation/mg protein is expressed as a percentage of the control. In unpaired Student's t-tests, control and GFP do not differ; Pixie, eIF4A and emetine are significantly lower than control, *P<0.0001. Depletion of Pixie and the translation initiation factor eIF4A was confirmed by western blotting and immunofluorescence (see Fig. S1 in the supplementary material; data not shown). Depletion of eIF4A or Pixie reduced translation by day 2, before any effect on cell number (data not shown) or cell cycle profile. Lower panels show cell cycle profiles of pixie RNAi-treated (thick line) and GFP RNAi control (thin line) cells obtained through FACS analysis for one of the above experiments. By day three, pixie RNAi treatment increases the G1 population. (D) Western blots of various fractions of an 0.8 M sucrose cushion through which S2 cells lysates were spun. Lysate, nuclei-free lysate; top, fraction above the cushion; C, cushion; P100, pellet obtained in presence of 100 mM KCl; P500, pellet obtained in presence of 500 mM KCl. Akt is not detected in the ribosomal pellet, whereas Pixie is (but not in the presence of high salt levels).





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