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. 2013 Jun 17;288(30):21537–21546. doi: 10.1074/jbc.M113.472290

FIGURE 3.

FIGURE 3.

Cdc123 is sub-stoichiometric to eIF2 subunits and binds unassembled Gcd11. A, binding of Cdc123 to unassembled Gcd11. myc-tagged versions of Gcd11, Cdc123 and the eIF2B-subunit Gcd1 were immunoprecipitated and analyzed for interaction of eIF2 subunits and Cdc123. Protein levels in yeast WCE and anti-myc immunoprecipitates (α-myc IP) were determined by Western analysis. B, cellular abundance of Cdc123 and eIF2 subunits. To compare endogenous protein levels of Cdc123 and eIF2 subunits, ha3 epitope sequences were fused to the chromosomal copies of CDC123, GCD11 (encoding eIF2γ), and SUI2 (encoding eIF2α). Dilution series of whole-cell lysates were analyzed by Western blotting. C, direct binding of Cdc123 to Gcd11. A maltose-binding protein (mbp) fusion of Gcd11 and his6-Cdc123 were co-expressed in E. coli, and mbp-Gcd11 was affinity-purified on amylose beads. Levels of mbp-Gcd11 and his6-Cdc123 in E. coli WCE and mbp-affinity precipitates (mbp-AP) were determined by Western analysis.