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. 2019 Sep 16;294(52):19852–19861. doi: 10.1074/jbc.RA119.009931

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© 2019 Lauenstein et al.

Author's Choice—Final version open access under the terms of the Creative Commons CC-BY license.

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Figure 4. — BCAP engages in SH3 domain interaction and tyrosine phosphorylation-dependent SH2 domain interactions. A, HEK293T cells were transfected with Myc-BCAP, Myc-BCAP Y374F, FLAG-GRB2, and FLAG-CRKL. At 24 h post-transfection, cells were lysed and subjected to immunoprecipitation with anti-FLAG antibody. Precipitates were split and immunostained for precipitation of FLAG-GRB2, FLAG-CRKL, and Myc-BCAP. B, purified GST-tagged GRB2, GRB2 SH2, CRKL; C, p85 N-SH2, p85 C-SH3, p85 SH3; D, PLC-γ2 N-SH2, PLC-γ2 C-SH2, and PLC-γ2 SH3 were immobilized on GST resin. Purified BCAP and dephosphorylated BCAP were subsequently applied to the resin and GST-tagged bait proteins were eluted from the resin and analyzed on SDS-PAGE. E, domain arrangement of BCAP-interacting proteins.