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. 2006 May 10;34(9):2495–2507. doi: 10.1093/nar/gkl290

Figure 4.

Figure 4

RNAi of TbEIF4AI. Procyclic T.brucei cells were transfected with the p2T7-177 derived plasmid containing the TbEIF4AI gene. Transfected cells were selected after growth in the presence of phleomycin and RNA interference induced after tetracycline addition. At regular intervals, cellular growth was monitored by counting the number of viable cells, expression of TbEIF4AI assayed and total protein synthesis investigated by [35S]methionine incorporation. (A) Cell density of transfected cultures with and without tetracycline addition. (B) Western blot analysis of the time course. Note the various dilutions of total cell extract for comparison (1–1/32 cell equivalent—1 cell equivalent equals to 106 cells and was used in the various RNAi lanes). TbEIF4AI was detected with the affinity purified antisera and anti-BiP was used as a loading control. The same blot was probed with both antibodies. Equivalent extracts of cells transfected with the p2T7-177/TbEIF4AIII construct (see also Figure 5) were also used in the blot to monitor for TbEIF4AI levels. (C) [35S]methionine incorporation profile in transfected cells grown without tetracycline or 24 and 48 h after its addition. Total protein synthesis was estimated after RNAi for TbEIF4AI by incubating aliquots of the cells in the presence of [35S]methionine for 1 h followed by TCA precipitation, quantitation of the incorporated radioactivity or SDS–PAGE followed by autoradiography of the selected samples.