Figure 5.
eEF1A is necessary for cytoplasmic retention of AID. (A) UV absorbance profile of total extracts from Ramos B cells treated for 2 h with vehicle, 2.5 µM CHX, 100 nM DidB, or 1 µM CytA, and fractionated by sucrose gradient. One representative out of three independent experiments is shown. (B) Representative confocal microscopy images (left) and nuclear signal quantitation (right) of Ramos B cells stably expressing AID-GFP treated for 2 h with either vehicle or increasing concentrations of DidB (top), CytA (middle), or CHX (bottom), as indicated. Plots (right graphs) indicate the quantitated nuclear signal in >40 individual cells per point, from 1 out of 2 independent experiments. Mean values are shown as black horizontal bars, and the mean value of untreated cells is shown as a blue line across the plot. (C) Ramos B cells stably expressing AID-GFP were treated for 24 h with DMSO, 10 nM CHX, or 1 nM DidB, and the nuclear GFP signal was quantified as in (B, left). One out of two independent experiments is shown. The mean fluorescence intensity (MFI) of AID-GFP measured by flow cytometry and normalized to DMSO + SD was plotted (right) for three independent experiments. Differences relative to DMSO were analyzed by ANOVA with Dunnett’s post-test (*, P < 0.05; **, P < 0.005; ****, P < 0.0001). (D) Representative confocal microscopy images are shown (left) of endogenous AID (detected by IF) in Ramos B cells after DMSO or 100 nM DidB treatment for the indicated times. Plots show nuclear signal quantified as in B from one out of two independent experiments (right). (E) Total cell extracts from HeLa cells transiently expressing AID and GFP or GFP-eEF1A1 and treated for 4 h with either vehicle, 2.5 µM CHX, 100 nM DidB, or 1 µM CytA, were immunoprecipitated with anti-GFP microbeads and analyzed by Western blot or by Urea-PAGE, stained with SYBR Gold to detect tRNA. One representative out of three independent experiments is shown. The mean binding of GFP-eEF1A1 to AID or tRNA normalized to DMSO (measured by densitometry) is indicated under the relevant blots. (F) Structure of the eEF1A homologue EF-Tu (33% identical to eEF1A1) in the GDP- (PDB:1TUI) and GTP-bound forms (PDB:1TTT, drawn with and without the tRNA molecule (gray)). The domains of EF-Tu (I, red; II, blue; III, green), and bound nucleotide (teal) are indicated. Bars, 10 µm.