Figure 1.
Figure 1. Association of DOCK2 with ELMO1 through its SH3 domain. (A) Schematic representation of DOCK2- and ELMO1-deletion mutants used in this study. The closed or hatched box indicates SH3 domain of DOCK2 or PH-like domain of ELMO1, respectively. (B) Total cellular RNA (10 μg) or polyA RNA (2 μg) was prepared from BW5147α–β– (lane 1), BW5147 (lane 2), BEα16-3 (lane 3), N3-5 (lane 4), 70Z/3 (lane 5), WEHI231 (lane 6), L1210 (lane 7), M12C3 (lane 8), J558 (lane 9), and NS1 (lane 10), and hybridized with DOCK2 or ELMO1 cDNA probe. (C) After either ELMO1 (lane 1), CrkL (lane 2), or empty vector (lane 3) was expressed with DOCK2 (lanes 1-3) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1 and CrkL) or anti-HA antibody (to detect DOCK2). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown. IP indicates immunoprecipitation; WB, Western blot. (D) After either DOCK2 (lane 1), DOCK2delC (lane 2), DOCK2delN (lane 3), DOCK2N (lane 4), or empty vector (lane 5) was expressed with ELMO1 (lanes 1-5) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2 or its deletion mutants) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1) or anti-HA antibody (to detect DOCK2 or its deletion mutants). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown. (E) After either DOCK2 (lane 1), DOCK2L27E (lane 2), DOCK2G32E (lane 3), DOCK2P60E (lane 4), or DOCK2F63E (lane 5) was expressed with ELMO1 (lanes 1-5) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2 or its SH3 mutants) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1) or anti-HA antibody (to detect DOCK2 or its SH3 mutants). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown. (F) After DOCK2 (lanes 2-5) or empty vector (lane 1) was expressed with ELMO1 (lanes 1-2), ELMO1del10 (lane 3), ELMO1del8 (lane 4), or ELMO1del1 (lane 5) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1 or its deletion mutants) or anti-HA antibody (to detect DOCK2). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown.

Association of DOCK2 with ELMO1 through its SH3 domain. (A) Schematic representation of DOCK2- and ELMO1-deletion mutants used in this study. The closed or hatched box indicates SH3 domain of DOCK2 or PH-like domain of ELMO1, respectively. (B) Total cellular RNA (10 μg) or polyA RNA (2 μg) was prepared from BW5147α–β– (lane 1), BW5147 (lane 2), BEα16-3 (lane 3), N3-5 (lane 4), 70Z/3 (lane 5), WEHI231 (lane 6), L1210 (lane 7), M12C3 (lane 8), J558 (lane 9), and NS1 (lane 10), and hybridized with DOCK2 or ELMO1 cDNA probe. (C) After either ELMO1 (lane 1), CrkL (lane 2), or empty vector (lane 3) was expressed with DOCK2 (lanes 1-3) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1 and CrkL) or anti-HA antibody (to detect DOCK2). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown. IP indicates immunoprecipitation; WB, Western blot. (D) After either DOCK2 (lane 1), DOCK2delC (lane 2), DOCK2delN (lane 3), DOCK2N (lane 4), or empty vector (lane 5) was expressed with ELMO1 (lanes 1-5) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2 or its deletion mutants) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1) or anti-HA antibody (to detect DOCK2 or its deletion mutants). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown. (E) After either DOCK2 (lane 1), DOCK2L27E (lane 2), DOCK2G32E (lane 3), DOCK2P60E (lane 4), or DOCK2F63E (lane 5) was expressed with ELMO1 (lanes 1-5) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2 or its SH3 mutants) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1) or anti-HA antibody (to detect DOCK2 or its SH3 mutants). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown. (F) After DOCK2 (lanes 2-5) or empty vector (lane 1) was expressed with ELMO1 (lanes 1-2), ELMO1del10 (lane 3), ELMO1del8 (lane 4), or ELMO1del1 (lane 5) in 293T cells, cell extracts were immunoprecipitated with anti-HA affinity matrix (to precipitate DOCK2) and analyzed by immunoblotting with the use of anti-V5 antibody (to detect ELMO1 or its deletion mutants) or anti-HA antibody (to detect DOCK2). The reactivity to anti-V5 antibody before (top panel) or after (middle panel) immunoprecipitation, and the reactivity to anti-HA antibody after immunoprecipitation (bottom panel) are shown.

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