Figure 3.

Anti-CD3 stimulation activates RAN for PLCβ3 exportation from the nucleus in Jurkat T-cells. A, the active form of RAN (RAN-GTP) in Jurkat T-cells upon anti-CD3 stimulation. Cells were stimulated for the indicated times with 10 μg ml⁻¹ anti-CD3. B, the active form of RAN (RAN-GTP) in Jurkat T-cells with ORP4L knockdown upon anti-CD3 stimulation. Cells were stimulated for 3 min with 10 μg ml⁻¹ anti-CD3 for 3 min. The relative GTP-RAN protein content was quantified from Western blots and normalized with the β-actin signal. C and D, Jurkat T-cells were stimulated for the indicated times with 10 μg ml⁻¹ anti-CD3 (C) or preincubated for 2 h with 50 ng ml⁻¹ LMB (D) followed by 5-min stimulation with 10 μg ml⁻¹ anti-CD3, and PLCβ3 phosphorylation at Ser⁵³⁷ was analyzed by Western blotting. E, confocal microscopy analysis of PLCβ3 and p-PLCβ3 in Jurkat T-cells after 5-min anti-CD3 stimulation (10 μg ml⁻¹) in the presence or absence of LMB. Scale bars, 10 μm. The data represent mean ± S.D. from an experiment performed in triplicate. **, p < 0.01; ***, p < 0.001, Student's t test. All error bars represent S.D. NT, nontargeting.