![Fig. 6. Localization of the CrkL:CD34ifullinteraction domain on CrkL. / To localize the putative CrkL domain that serves to interact with CD34, GST-CrkL fusion proteins containing partial CrkL sequences were constructed and then used to precipitate endogenous CD34 protein. (A) GST-CrkL fusion proteins. The native CrkL protein contains 1 SH2 and 2 SH3 domains, as shown. Depicted are the CrkL constructs used in studies designed to precipitate interacting proteins, including CD34. Two GST-CrkL fusion proteins were constructed: GST-5′CrkL encompassed CrkL amino acids 1 to 194, covering the SH2 and N-terminal SH3 domains (but not the C-terminal SH3); GST-3′CrkL encompassed CrkL amino acids 197 to 303, covering the C-terminal SH3 only. (B) GST-CrkL precipitation studies. KG1a cells were stimulated 5 minutes with 9C5 antibody (“CD34”) to engage the extracellular domain of CD34 in adhesion assays (or with control immunoglobulin MOPC 21 [“Ig”]). Lysates from 4 × 106 cells per point were precleared with GST-Sepharose, and then proteins were pulled down with 20 μg GST, 20 μg GST-CD34ifull (“CD34i”), 2 μg GST-3′CrkL C-terminal SH3 domain (“CrkL 3′SH3”), or 2 μg 5′CrkL SH2–N-terminal SH3 domain (“CrkL 5′SH2-SH3”) bound to GST-Sepharose. Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted (WB) with QBEND10 CD34 antibody. Results demonstrate constitutive association between endogenous CD34 and the C′SH3 domain of GST-CrkL protein but not to the SH2-N′SH3 CrkL domain. In contrast, the SH2-N′SH3 CrkL domain, but not the C′SH3 domain, precipitated C-Abl (Figure 7B).](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/97/12/10.1182_blood.v97.12.3768/6/h81211161006.jpeg?Expires=1725169840&Signature=N5WKi7IZiT7nuKFR021ogaTPuaIKoUyCRynX4vBO6EezFL7AKQFSNyjNle3liEDCkF0lqcDjzAmTU77wfN8CGyimvxijS31SaktkgGC-07HtbLot85K1TZFbxyF0wY~lq4k16BdMzN8iVbo3zbkpVt2b3MBiuqb47qUK9MQpea4iaGNyTCyuhEwZXIOIP2Sl6lwtmVGXZGVNc7LfAyEl11HAffv0Rcu36wjz61op8f5jKFubsEDmR2APQM5DnkRiaJMUjDQthD7jcbT94qArn1jdMy2KAh5-~eEWIzcm5zAnXc9jcjtRnR0Ff94rkgbRiUrDCxGHIFjAwfQVxCXbyg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Localization of the CrkL:CD34ifullinteraction domain on CrkL.
To localize the putative CrkL domain that serves to interact with CD34, GST-CrkL fusion proteins containing partial CrkL sequences were constructed and then used to precipitate endogenous CD34 protein. (A) GST-CrkL fusion proteins. The native CrkL protein contains 1 SH2 and 2 SH3 domains, as shown. Depicted are the CrkL constructs used in studies designed to precipitate interacting proteins, including CD34. Two GST-CrkL fusion proteins were constructed: GST-5′CrkL encompassed CrkL amino acids 1 to 194, covering the SH2 and N-terminal SH3 domains (but not the C-terminal SH3); GST-3′CrkL encompassed CrkL amino acids 197 to 303, covering the C-terminal SH3 only. (B) GST-CrkL precipitation studies. KG1a cells were stimulated 5 minutes with 9C5 antibody (“CD34”) to engage the extracellular domain of CD34 in adhesion assays (or with control immunoglobulin MOPC 21 [“Ig”]). Lysates from 4 × 106 cells per point were precleared with GST-Sepharose, and then proteins were pulled down with 20 μg GST, 20 μg GST-CD34ifull (“CD34i”), 2 μg GST-3′CrkL C-terminal SH3 domain (“CrkL 3′SH3”), or 2 μg 5′CrkL SH2–N-terminal SH3 domain (“CrkL 5′SH2-SH3”) bound to GST-Sepharose. Precipitated proteins were separated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted (WB) with QBEND10 CD34 antibody. Results demonstrate constitutive association between endogenous CD34 and the C′SH3 domain of GST-CrkL protein but not to the SH2-N′SH3 CrkL domain. In contrast, the SH2-N′SH3 CrkL domain, but not the C′SH3 domain, precipitated C-Abl (Figure 7B).
