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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 1. Scd6 binds eIF4G.

Figure 1

A) 32P labeled MFA2 mRNA was incubated with GST, GST-Scd6 or GST-Scd6ΔRGG followed by pull down using glutathione sepharose. Radioactivity on beads after three washes was measured by Cerenkov counting. B) Purified His-Pab1 was incubated with FLAG-Scd6 followed by FLAG pull down. Pab1 and Scd6 were detected by using polyclonal anti-Pab1 and anti-Scd6 antibodies respectively. C) Purified Scd6 and RGG mutant were tested for their effects on translation of 100 ng of poly(A−) and poly(A+) luciferase message at 6uM concentration as described previously (Nissan et al., 2010). D) Glutathione sepharose pull downs performed to look at interaction of purified GST-4G/E with recombinant Scd6 and Scd6ΔRGG mutant protein. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. E) Glutathione sepharose pull downs were performed to look at interaction of GST-4G in bacterial extracts with recombinant Scd6 and Scd6ΔRGG mutant protein. 6ug of each protein was used in 200ul reaction mixture. Scd6 protein was detected with anti-FLAG antibody following manufacturer’s instructions. Typically 10% of total reaction was loaded in the ‘input’ lanes and 100% of total pulled-down material was loaded in ‘pellet’ lanes.