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. 2011 Jul 14;39(19):8404–8415. doi: 10.1093/nar/gkr555

Figure 5.

Figure 5.

Leish4E-IP interacts with LeishIF4E-1 and LeishIF4E-4 in promastigotes. Pull-down analysis with SBP tagged LeishIF4E-1, LeishIF4E-4 or Leish4E-IP from L. amazonensis was done using affinity purification and analysis as described in Figure 1A. Aliquots of the soluble extract (S, 1%), the flow-through (F, 1%), the final wash (W, 40% for A, C, D and 20% for B) and eluted proteins (E, 40% for A, C, D and 20% for B) were separated by SDS–PAGE (10–15%) and subjected to western blot analysis using specific antibodies against LeishIF4E-1, LeishIF4E-4, Leish4E-IP and SBP. The pull-down analysis was performed in promastigotes (A and B), promastigotes after 2 h at 33°C (C) and in axenic amastigotes, 9 days after differentiation (D) Densitometric analysis of panel A showed that the elution fraction contained 10.4 and 14% of LeishIF4E-1 and Leish4E-IP, respectively (top) and 8 and 12% of LeishIF4E-4 and Leish4E-IP, respectively (bottom). The wash fraction of Leish4E-IP that was pulled down by LeishIF4E-4 contained 7% of total protein, suggesting that the eluted fraction contained some protein that came down non-specifically.