Fig 2: CBAP is important for chemokine-dependent activation of Vav1 and Rac1 in T cells.Analysis of chemokine-induced activation of ZAP70 and Vav1 in (Ai) Jurkat T cells and (Aii) primary T cells. Cells were stimulated with CXCL12 (200 ng/ml) at the indicated times. Total cell lysates were subjected to immunoblotting with antibodies as indicated. (B) CBAP-knockdown (C29) cells transfected with a plasmid encoding enhanced GFP (vec) or GFP-tagged murine CBAP (mCBAP-GFP) were stimulated with CXCL12 and subjected to immunoblotting with antibodies as indicated. (C) Effect of piceatannol treatment on CBAP-dependent Vav1 phosphorylation. After a 3-h incubation with DMSO or piceatannol (25 µM), cells were stimulated with CXCL12 and subjected to immunoblotting with antibodies as indicated. The numbers indicated below the pVav1 blots are presented as the fold change over the control bands (B, C). (D) CXCL12-induced activation of Rac1 was analyzed by PBD-pulldown assay (see Materials and Methods). C21 and C29 Jurkat T-cell clones were stimulated with CXCL12 for the indicated times. Total cell lysates were prepared and pulled down with GST-PBD and subjected to immunoblotting to detect Rac1 (upper panel). Each total cell lysate was also blotted directly with anti-Rac1 as a loading control (lower panel). Quantified data are shown below the blot as relative intensity of Rac1-GTP compared to that at the zero time point from the same samples of three independent experiments. (E) Activation of Rac1 in CBAP -/- primary T cells was analyzed after stimulation with CXCL12 (200 ng/ml) or CCL21 (200 ng/ml). CBAP +/+ and CBAP -/- T cells purified from LNs were stimulated with CXCL12 or CCL21 for the indicated times. Total cell lysates were analyzed, and the activity of Rac1-GTP is presented as described in (D). (F, G) Effect of piceatannol treatment on the migration and adhesion of CBAP-knockdown Jurkat clones. DMSO- or piceatannol (pic)-pretreated cells were stimulated with CXCL12 and analyzed for cell migration (F) and adhesion (G) as described in the legends to Figure 1B and 1E, respectively. The data presented represent the mean ± SD of five individual experiments. *P<0.05 and ***P<0.005.

Fig 3: Complex formation among CBAP, ZAP70 and integrin ß1.(A) Schematic representation of GFP-tagged full-length (FL) and various deletion mutants (M1–M5) of CBAP. aa, amino acid. (B, C, D) Cell lysates from 293T cells co-expressing various combinations of the indicated proteins were immunoprecipitated (IP) with anti-GFP followed by immunoblotting (IB) using the indicated antibodies. As input controls, total cell lysates (1/100) were also blotted with the relevant antibody, and the results are shown at the bottom of each panel. (E) A proposed model for the role of CBAP in chemokine-induced ZAP70-mediated dissociation of the Vav1-talin complex (see text for details). Components in Black-and-white have been established previously [10], and Components in color were established in the current study.