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. Author manuscript; available in PMC: 2012 Jul 27.
Published in final edited form as: Mol Cell. 2012 Jan 27;45(2):244–254. doi: 10.1016/j.molcel.2011.11.026

Figure 3. Scd6 does not destabilize Pab1-4G or 4A-4G interactions.

Figure 3

A) 7-methyl-GTP sepharose pull downs were performed to look at interaction of GST-eIF4G/eIF4E complex with recombinant Pab1 in presence of Scd6 or Scd6ΔRGG protein as described in materials and methods. 8ug of GST-eIF4G/eIF4E was incubated with wt or mutant Scd6 protein for 1h at 4°C with end-to-end rotation. Following this Pab1 was added to reaction mixture and incubated for 1h with end-to-end rotation. Finally, 7-methyl-GTP sepharose was added to reaction mix and incubated for 2h. Washing was performed as described in materials and methods. eIF4G and Pab1 were detected with polyclonal antibodies. B) Glutathione pull down assay to analyze effect of purified Scd6 on eIF4A-eIF4E/G interaction. Incubation and washing conditions were same as mentioned in A. eIF4G and eIF4A were detected with polyclonal antibodies. The anti-eIF4A antibody cross-reacts with purified Scd6 (contains both His and FLAG-tag). The middle panel depicts ponceau-stained blot with clearly visible wt and mutant Scd6 proteins. This was done since eIF4A and Scd6ΔRGG run at similar position (in top panel). Typically 10% of total reaction was loaded in the ‘input’ lanes and 50 or 100% of total pulled-down material was loaded in ‘pellet’ lanes.