FIG. 4.
Dph3 is required for diphthamide modification on eEF-2. (A) eEF-2 of dph3−/− embryos is resistant to ADP ribosylation by DT. Lysates from different dph3 genotype E10.5 embryos were incubated with [32P-adenylate]NAD in the absence or presence of DT. Reactions were terminated by boiling in SDS sample buffer, and the samples were analyzed by SDS-PAGE followed by autoradiography. (B) One allele of dph3 is sufficient for diphthamide modification of all eEF-2 in mouse. Lysates from ES cells and dph3−/−, dph3+/−, and dph3+/+ embryos were incubated with DT at room temperature for 30 min, and the lysates were then analyzed by native PAGE followed by Western blotting using an antibody against a linear peptide of the carboxyl terminus of eEF-2.