FIGURE 1.
TRAF6 interacts with Vav3. A, TRAF6 interacts with Vav3 in vitro. Left panels, 293T cells were transfected with GST-TRAF6 (1.0 μg) with or without Xp-Vav3 (1.0 μg). The cell lysates were normalized for total protein content. GST-TRAF6 was pulled down (PD) using GST beads, and the PD GST-TRAF6 was visualized via anti-GST (B-14) immunoblotting (IB, middle panel). Vav3 bound to the TRAF6 was visualized via anti-Xp IB (top panel). The level of Vav3 expression was detected via anti-Xp IB (bottom panel). GST alone was used as a control. Right panels, Vav3-Myc was immunoprecipitated (IP) using anti-Myc beads. TRAF6 bound to Vav3 was visualized via anti-FLAG (M2) IB (top panel). B, identification of the endogenous TRAF6-Vav3 interaction. Endogenous TRAF6 was IP with an anti-(α)-TRAF6 (D-10) antibody. The cell lysates were normalized for total protein content. Protein bound to TRAF6 was visualized via anti-Vav3 IB. β-Actin was used as a loading control. C, subcellular localization of TRAF6 and Vav3. 293T cells were transfected with Vav3-Myc and TRAF6-RFP. The transfected cells were immunostained with an anti-Myc (9E10) antibody and a FITC-labeled secondary antibody, and the subcellular localization of TRAF6 (red) and Vav3 (green) was visualized by confocal microscopy (magnification, ×800). D, TRAF6 interacts with Vav3 in RANKcyto. 293T cells were transfected with GST-RANKcyto (0.8 μg) and Vav3-Myc (0.4 μg) with or without FLAG-TRAF6 (0.3–0.9 μg). The cell lysates were normalized for total protein content. Proteins bound to GST-RANKcyto were visualized via anti-Myc IB (top panel) or anti-FLAG IB (second panel). E, identification of Vav3 interactions with other TRAF members recruited to RANKcyto. 293T cells were co-transfected with FLAG-TRAFs (1.0 μg) and Vav3-Myc (1.0 μg). The cell lysates were normalized for total protein content. Vav3 bound to TRAF was visualized via anti-Myc IB (top panel). All experiments were performed at least three times with similar results.