FIGURE 2.

LIF stimulation activates ALR3 and enhances ARL3-STAT3 interaction. A, 293T cells (1 × 10⁷) were treated with LIF (100 ng) for 30 min. Cells were lysed and mixed with GST or GST-BART protein (10 μg). The mixture was then pulled down with glutathione-Sepharose beads, followed by immunoblotting (IB) with anti-ARL3 or anti-GST antibodies. An aliquot of TCL (1%) was blotted with anti-ARL3 antibody or anti-pSTAT3 antibody. B, HeLa cells (1 × 10⁷) were transfected with HA-STAT3 WT (10 μg) with or without Myc-ARL3 (10 μg). At 48 h after transfection, the cells were lysed and mixed with GTPγS. The immunoprecipitate (IP) with anti-Myc was then blotted with anti-HA or anti-Myc antibodies. TCL (1%) was blotted with anti-HA or anti-Myc antibody. C, HeLa cells in a 6-well plate were transfected with empty vector, Myc-tagged ARL3 WT, ARL3 QL, or TN (2 μg). The cells were stimulated with LIF (100 ng/ml) for 30 min or IL-6 (10 ng/ml) for 60 min. The activities of the GTPase cycle were calculated from luciferase activity of the lysate. *, p < 0.05. D, 293T cells (1 × 10⁷) were transfected with HA-STAT3 WT (WT) with or without Myc-ARL3 WT, QL, or TN (10 μg). 48 h after transfection, the cells were lysed and immunoprecipitated with anti-Myc, followed by immunoblotting with anti-HA or anti-Myc antibodies. TCL (1%) was blotted with anti-Myc antibody. E, 293T cells in a 24-well plate were transfected with STAT3-LUC (100 ng) with or without ARL3 WT, ARL3 QL or ALR3 TN (100 ng). At 36 h after transfection, cells were treated with LIF (100 ng/ml) for an additional 12 h. The cells were harvested and assayed for luciferase activity. The results are indicated as fold induction of luciferase activity from triplicate experiments, and the error bars represent S.D. F, 293T cells in a 24-well plate were transfected with STAT3-LUC (100 ng) with or without STAT3-C and ARL3 WT, ARL3 QL, or ALR3 TN (100 ng). At 48 h after transfection, cells were harvested and assayed for luciferase activity as described above. **, p < 0.01. G, 293T cells in a 24-well plate were transfected with STAT3-LUC (100 ng) with empty vector, ARL3 WT, ARL3 QL, or ALR3 TN (100 ng). Relative luciferase activity represents the ratio of luciferase to β-galactosidase activity. Shown is a representative experiment, which was repeated at least three times with similar results.