Figure 2.

YB-1 regulates G3BP1 mRNA translation. (A) U2OS cells transfected with siControl or siYB-1 siRNAs were treated with vehicle alone, arsenite (0.5 mM), or H₂O₂ (0.5 mM) for 1 h. Lysates were analyzed by immunoblotting using the indicated antibodies. (B) Band intensities from A were quantified by densitometry. Mean values ± SD (error bars) are shown for three independent experiments. **, P < 0.01. (C) Total RNA isolated from siControl and siYB-1 U2OS cells under ambient conditions was subjected to RT-PCR using primers for YB-1 and G3BP1. Mean values ± SD (error bars) are shown for three independent experiments. ns, nonsignificant. (D) U2OS cells transfected with siControl or siYB-1 siRNAs were incubated with CHX for the indicated times, and lysates were immunoblotted using the indicated antibodies. (E) U2OS cells transfected with siControl or siYB-1 siRNAs were treated with vehicle alone, arsenite (0.5 mM), or H₂O₂ (0.5 mM) for 1 h in the presence of AHA to capture newly synthesized proteins, and immunoblotted using the indicated antibodies. (F) Band intensities from E were quantified using densitometry. Mean values ± SD (error bars) are shown for three independent experiments. **, P < 0.01. (G) siControl or siYB-1 kd U2OS cells were transfected with Myc-G3BP1 (G3BP1 overexpression [OE]), and treated with vehicle alone (VA) or arsenite (AR; 0.5 mM) for 1 h. Lysates were then prepared and immunoblotted using antibodies against G3BP1, YB-1, and GRB2. (H) The same cells were fixed and subjected to IF using the indicated antibodies. SGs were quantified as in Fig. 1 B. Mean values ± SD (error bars) are shown for three independent experiments. **, P < 0.01. ns, nonsignificant. Bars, 10 µm.