1. DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines
by Hiroshi Nishihara, Masae Maeda, Atsushi Oda, Masumi Tsuda, Hirofumi Sawa, Kazuo Nagashima, and Shinya Tanaka
Blood
Volume 100(12):
December 1, 2002
©2002 by American Society of Hematology

2. The association of DOCK2 and GST-CrkL-SH3(N)
The association of DOCK2 and GST-CrkL-SH3(N).(A) Lysates of human normal platelets were subjected to pull-down assay by GST (lane 1) or GST-CrkL-SH3(N) (lane 2).
The association of DOCK2 and GST-CrkL-SH3(N).(A) Lysates of human normal platelets were subjected to pull-down assay by GST (lane 1) or GST-CrkL-SH3(N) (lane 2). The precipitates were analyzed by immunoblotting with anti-DOCK2 (αDOCK2) Ab. Arrowhead indicates the size of DOCK2. (B) Lysates of Jurkat and MOLT4 were subjected to pull-down assay by GST (lanes 2 and 5) or GST-CrkL-SH3(N) (lanes 3 and 6). Arrowhead indicates the size of DOCK2. (C) Analysis of the binding of DOCK2 and various SH3 domains (indicated at the top) by pull-down assay in Jurkat cells lysates. The precipitates were analyzed by immunoblotting by means of anti-DOCK2 Ab (top panel) and anti-GST Ab (bottom panel). Lane 1, pull-down (−) indicates total cell lysates. In the bottom panel, asterisks indicate the expected size of each SH3 domain–containing proteins.
Hiroshi Nishihara et al. Blood 2002;100:
©2002 by American Society of Hematology

3. The association of DOCK2 and CrkL in vivo
The association of DOCK2 and CrkL in vivo.(A) Analysis of the association of CrkL with DOCK2 in 293T cells.
The association of DOCK2 and CrkL in vivo.(A) Analysis of the association of CrkL with DOCK2 in 293T cells. The pCAGGS-CrkL and pCXN2-Flag-DOCK2 were transiently transfected, and an immunoprecipitation assay was performed by anti-CrkL Ab (lane 2) or normal rabbit serum (NRS; lane 1). Immunoblotting was performed with the use of anti-Flag Ab (top panel) and anti-CrkL Ab (bottom panel). Total cell lysates appear in lane 3. (B) Analysis of the association of c–Crk-II with DOCK2 in 293T cells. The sets of cotransfected plasmid were as follows: pCAGGS–c–Crk-II and pCXN2 vector (lanes 1, 3, and 4); pCAGGS–c–Crk-II and pCXN2-Flag-DOCK2 (lanes 2, 5, and 6). Cell lysates were analyzed by immunoprecipitation with the use of anti-DOCK2 (αDK2) Ab or NRS (lanes 3-6). Total cell lysates are shown in lanes 1 and 2. (C) Association of CrkL and DOCK2 in Jurkat cells with stimulation. Cells were in suspension (sus; lanes 1 and 2), plated on collagen type I–coated dishes (collagen; lanes 3 and 4), or plated on poly-L-lysine–coated dish (poly-L-K; lanes 5 and 6). Cells were incubated for 15 minutes at 37°C, and cell lysates were immunoprecipitated with anti-DOCK2 (αDK2) Ab (lanes 2, 4, and 6) or NRS (lanes 1, 3, and 5), and precipitates were analyzed by immunoblotting with antibodies, indicated below each panel. Total cell lysates appear in lane 7. The arrowheads indicate DOCK2 in the top panel, Vav in the second panel, CrkL in the third panel, and c–Crk-II in the bottom panel. (D) Association of endogenous CrkL and DOCK2 in Jurkat, MOLT4, and Raji cells. Cell lysates were immunoprecipitated by anti-CrkL Ab (αCrkL; lane 3, 9, and 13) or anti-DOCK2 Ab (αDK2; lane 6, 10, and 14) and probed with anti-DOCK2 Ab (top panels) and with anti-CrkL Ab (bottom panels).
Hiroshi Nishihara et al. Blood 2002;100:
©2002 by American Society of Hematology

4. Analysis of the association of CrkL with DOCK2 truncated mutants
Analysis of the association of CrkL with DOCK2 truncated mutants.(A) Schematic structure of wild-type DOCK2 and the mutants designated dN (aa 939 to 1830), dCS (aa 1 to 515), INT1 (aa 386 to 593), INT2 (aa 586 to 883), dBE (aa 926 to 1478), and CT (aa
Analysis of the association of CrkL with DOCK2 truncated mutants.(A) Schematic structure of wild-type DOCK2 and the mutants designated dN (aa 939 to 1830), dCS (aa 1 to 515), INT1 (aa 386 to 593), INT2 (aa 586 to 883), dBE (aa 926 to 1478), and CT (aa 1640 to 1830). ND indicates not determined; P, proline-rich sequence; B, basic domain; and S, SH3 domain. (B) Association of CrkL and DOCK2 mutants were analyzed by immunoprecipitation. The 293T cells were transfected with pCAGGS-CrkL and the DOCK2 expression vector, and cell lysates were immunoprecipitated with anti-CrkL Ab and were analyzed by immunoblotting with anti-Flag monoclonal Ab (Ab; lanes 6-10). Protein expressions of DOCK2 mutants and CrkL were examined by anti-Flag mAb (top panel, lanes 1-5) and anti-CrkL Ab (bottom panel). Transfected plasmids were as follows: lanes 1 and 6, pCAGGS; lanes 2 and 7, pCXN2-Flag-DOCK2-dCS; lanes 3 and 8, pCXN2-Flag-DOCK2-dN; lanes 4 and 9, pCXN2-Flag-DOCK2-dBE; and lanes 5 and 10, pCXN2-Flag-DOCK2-CT.
Hiroshi Nishihara et al. Blood 2002;100:
©2002 by American Society of Hematology

5. Analysis of the activation of Rac1 by pull-down assay using PAK2-RBD
Analysis of the activation of Rac1 by pull-down assay using PAK2-RBD.(A) Activation of endogenous Rac1 in Jurkat and K562 cells.
Analysis of the activation of Rac1 by pull-down assay using PAK2-RBD.(A) Activation of endogenous Rac1 in Jurkat and K562 cells. Jurkat and K562 cells were transfected with either empty vector or pCXN2-Flag-DOCK2, and drug-resistant cells were lysed and incubated with GST-PAK2-RBD and glutathione-Sepharose beads. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-Rac1 mAb (top and right panels). Expression of DOCK2 proteins was examined by immunoblotting by anti-Flag mAb (right panels). The intensities of precipitated Rac1 were measured and are depicted as a bar graph comparing each with the value of vector control as 1.0. (B-D) The 293T cells were transfected with pCXN2-Flag-Rac1. Expression vectors for the proteins are indicated at the bottom. Cells were lysed and incubated with GST-PAK2-RBD and glutathione-Sepharose beads. The bound proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-Flag mAb (top panel and bottom graph). Expression of transfected proteins was examined by immunoblotting by anti-Flag mAb for Vav, DOCK2, and DOCK180, and by anti-CrkL Ab (right panels). The intensities of precipitated Rac1 were measured and are depicted as a bar graph comparing each with the value of vector control as 1.0. Panel B shows activation of Rac1 by CrkL, Vav, DOCK2, DOCK180; panel C, activation of Rac1 by DOCK2 and its truncated mutants; and panel D, suppression of CrkL- and Vav-induced Rac1 activation by the DOCK2 dCS mutant.
Hiroshi Nishihara et al. Blood 2002;100:
©2002 by American Society of Hematology

6. Colocalization of DOCK2 with CrkL and F-actin in the hematopoietic cell line.Jurkat cells were transfected with expression vectors of both CrkL and DOCK2 (panels Aiv-v), of DOCK2 (panels Biv-v), or vector alone (panels Ai-ii,Biv-v), and at 24 hours after tr...
Colocalization of DOCK2 with CrkL and F-actin in the hematopoietic cell line.Jurkat cells were transfected with expression vectors of both CrkL and DOCK2 (panels Aiv-v), of DOCK2 (panels Biv-v), or vector alone (panels Ai-ii,Biv-v), and at 24 hours after transfection, cells were plated on fibronectin-coated slides and fixed with paraformaldehyde. Localization of CrkL and DOCK were analyzed by anti-CrkL (panels Aii,v) and anti-Flag Abs (panels Ai,iv,Bii,v), respectively. F-actin was visualized by phalloidin conjugated with Alexa-594 (panels Bi,iv). Cells were observed with laser scanning confocal microscopy, and merged images are displayed (panels Aiii,vi,Biii,vi). Original magnification × 400.
Hiroshi Nishihara et al. Blood 2002;100:
©2002 by American Society of Hematology

7. Establishment of Jurkat cell line stably expressing DOCK2 and DOCK2-dCS.(A) Expression levels of DOCK2 of proteins were examined by immunoblotting with anti-Flag mAb.
Establishment of Jurkat cell line stably expressing DOCK2 and DOCK2-dCS.(A) Expression levels of DOCK2 of proteins were examined by immunoblotting with anti-Flag mAb. Lane 1, parental Jurkat cells; lane 2, Jurkat cells with vector control; lane 3, Jurkat cells with DOCK2-WT; lane 4, Jurkat cells with DOCK2-dCS. (B) Measurement of active form of Rac1 using established cell lines. For the positive control, Jurkat cells were preincubated with phorbol 12-myristate 13-acetate (PMA; Sigma) (1 μM) for 10 minutes. Cells were lysed and incubated with GST-PAK2-RBD and glutathione-Sepharose beads. The bound proteins were separated by SDS-PAGE, and endogenous Rac1 was detected by immunoblotting with anti-Rac1 mAb (B&D Transduction Laboratories). The intensities of precipitated Rac1 were measured and are depicted as a bar graph comparing each with the value of nontransfected control as 1.0. (C) Assessment of cell adhesion. Cells were plated on collage type I–coated (top panels) or poly-L-lysine–coated (bottom panels) dishes, and incubated at 37°C for 20 minutes. Cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature and observed by phase contrast microscopy. Original magnification × 400.
Hiroshi Nishihara et al. Blood 2002;100:
©2002 by American Society of Hematology