Fig. 3From: eEF1Bγ binds the Che-1 and TP53 gene promoters and their transcriptsCharacterization of the eEF1Bγ mRNA binding property a Polysome profiles of HeLa cells by sedimentation velocity through sucrose density gradients. Cytoplasmic extracts were untreated (NT) or treated with 100 mM EDTA to dissociate polyribosomes. Representative absorbance profiles at 254 nm. The 80S monosome peak is indicated (right). Western blot analysis showing the distribution of the eEF1Bγ protein in fractions collected from the top to the bottom of the sucrose gradient. Samples corresponding to mRNPs, 40S, 60S, 80S monosomes and polysomes fractions are indicated. SMN and the ribosomal proteins S6 and L7 were used as controls (left). b Schematic representations of myc-tagged full-length eEF1Bγ and its derived deletion mutants transiently transfected in HeLa cells analyzed in panel c. c RIP assays were performed with an anti-myc tag antibody. Immunoprecipitated samples were analyzed by western blotting using the myc-tag monoclonal antibodies to verify IP efficiency. The asterisks mark the signal corresponding to the eEF1Bγ mutant, partially covered by the heavy chain Ig band (top) and a non-specific band (bottom). The total cell lysates were immunoblotted to verify the correct expression of the transfected molecules (left). On the right, the graph shows the analysis of mRNAs immunoprecipitated with the indicated constructs. The data are expressed as percent precipitation relative to input mRNAs. The horizontal line illustrates the mean background levelBack to article page