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

FIGURE 4.

FIGURE 4.

The PH domain of P-Rex1 is required for Norbin binding. A, schematic of P-Rex1 mutants. B, purified mutant P-Rex1 proteins. Coomassie-stained SDS-PAGE showing 100 pmol of recombinant full-length or mutant EE-P-Rex1 proteins (50 pmol for EE-IP4P), purified from Sf9 cells. C, the PH domain of P-Rex1 is required for Norbin binding in vitro. 4.5 pmol of purified full-length or mutant EE-P-Rex1 proteins were incubated with 4.5 pmol of purified GST-Norbin or GST, or with beads alone, and Western blotted with EE antibody. Blots shown are from 1 experiment representative of 3 for the full-length protein and for ΔPH, ΔDEP, and ΔPDZ mutants and 5 for ΔIP4P and iIP4P. D, the PH domain of P-Rex1 is required for the interaction with Norbin in vivo. COS-7 cells expressing combinations of myc-Norbin full-length (FL) EE-P-Rex1, EE-ΔDEP, or EE-ΔPH were immunoprecipitated with immobilized EE antibody under stringent conditions (RIPA buffer) and analyzed by Western blotting with myc-HRP, EE, and P-Rex1 6F12 ABs. The white vertical lines show a cropped lane. Blots shown are from 1 experiment representative of 4.