Fig. 4.

eEF1A1 and eEF1A2 are phosphorylated at tyrosine residues in vivo. (A) 0.5 μg of purified eEF1A1 and eEF1A2 was immunobloted with anti-phosphotyrosine-specific antibodies (4G10) followed by WB with anti-eEF1A antibodies (upper panel). SDS-PAGE of purified eEF1A1 and eEF1A2 visualized by Coomassie staining, 1 μg of each protein was loaded per line (lower panel). (B) NiNTA sepharose beads with precipitated eEF1A1/eEF1A2 proteins were divided on two samples, the first was treated with 5 U CIAP at 37 °C for 1 h, second was incubated at 37 °C for 1 h in the presence of 1 mM NaOVa. NiNTA sepharose beads were washed with the lysis buffer two times; proteins were eluted by boiling in Laemmli sample buffer and resolved by SDS-PAGE. Phosphorylated eEF1A1 and eEF1A2 were visualized by immunoblotting with anti-phosphotyrosine-specific antibodies (4G10) (upper panel) followed by WB with anti-His tag antibodies (lower panel).