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. 2016 Jan 20;291(12):6359–6375. doi: 10.1074/jbc.M115.686592

FIGURE 3.

FIGURE 3.

Norbin binds P-Rex1 directly and interacts with P-Rex1 endogenously in cells. A, myc-Norbin interacts with EE-P-Rex1. COS-7 cells expressing EE-P-Rex1 and/or myc-Norbin were subjected to immunoprecipitation with immobilized EE antibody under stringent conditions (1% Triton X-100) and analyzed by Western blotting with myc and EE Abs as indicated. Blots shown are from 1 experiment representative of 3. B, endogenous P-Rex1 and Norbin interact. HEK-293 cell lysates were incubated with Norbin antibody pool (C1, N4, N7) or mock-treated with serum-IgG, washed stringently and Western blotted. Overexpression of myc-Norbin was used as a control. Blots shown are from 1 experiment representative of 3. C, purification of GST-Norbin. Coomassie gel showing purified recombinant GST-Norbin and GST (bead volumes are indicated) and BSA standards. The vertical white lines denote cropped lanes. D, direct interaction between EE-P-Rex1 and GST-Norbin. 10 pmol of immobilized purified recombinant GST-Norbin or GST, or beads alone, were incubated with 20 pmol of purified recombinant EE-P-Rex1, beads were washed stringently, and proteins were analyzed by Western blotting. Blots shown are from 1 experiment representative of 3.