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. 2000 Mar;20(5):1649–1658. doi: 10.1128/mcb.20.5.1649-1658.2000

FIG. 3.

FIG. 3

Association of CIZ with Cas. (A) FLAG-tagged CIZ expressed in COS-7 cells was precipitated with GST (lane 1), GST-Cas SH3 (lane 2), GST-Src SH3 (lane 3), GST-Fyn SH3 (lane 4), GST-Crk SH3 (lane 5), GST-Ash/Grb2 SH3 (lane 6), and GST-Abl SH3 (lane 7) and was immunoblotted with anti-FLAG (upper row). The samples were electrophoresed and stained with Coomassie blue (bottom row). Total lysates of COS-7 cells expressing CIZ-FLAG were also immunoblotted (lane 8). (B) FLAG-tagged CIZ (lanes 1 and 5), mPR mutant of CIZ-FLAG (lanes 2 and 6), and mock (lanes 3 and 7)-transfected COS-7 cell lysates were incubated with GST-Cas SH3 (lanes 1, 2, and 3) or with GST (lanes 5, 6, and 7) and immunoblotted with anti-FLAG. Lanes 4 and 8 contained GST-Cas SH3 or GST alone, respectively. vec, vector. (C) CIZ-FLAG was expressed in COS-7 cells, and the lysates were immunoprecipitated (IP) with anti-Cas2 (lane 1) or with preimmune (Pre) serum (lane 2). The total cellular lysates (TL) are shown in lane 3. The samples were immunoblotted with anti-Cas2 in the upper row and immunoblotted with anti-FLAG in the bottom row. (D) Association between CIZ and Cas expressed in COS-7 cells. CIZ-FLAG (lanes 2 and 6) or mPR-FLAG (lanes 1 and 5) was expressed in COS-7 cells together with Cas. Cas alone (lanes 3 and 7) and CIZ-FLAG alone (lanes 4 and 8) were also expressed. In lanes 1 to 4, the lysates were immunoprecipitated with anti-FLAG. In the upper row, the filter was immunoblotted with anti-Cas2. In the bottom row, the same immunoprecipitant was immunoblotted with anti-FLAG. (E) The wild-type Cas (wtCas) (lanes 1 and 3) and the mutant of Cas that lacks the SH3 domain (lanes 2 and 4 [dSH3]) were expressed together with FLAG-tagged CIZ. The lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-Cas2 (upper row) or anti-CIZ (lower row). (F) The lysates of LC-540 cells were immunoprecipitated with preimmune serum (lane 1), anti-CIZ (lane 2), and anti-Cas2 (lane 3). In the upper row, the filter was immunoblotted with anti-Cas2, and in the lower row, the filter was immunoblotted with anti-CIZ.