The N-terminal 95 (1 to 95) amino acids and 429 amino acids (1 to 429) of NBCe1-B were subcloned in the pQE-TriSystem His-Strep vector (Qiagen). The final plasmid constructs were transformed into the Escherichia coli strain Rosetta (DE3) competent cells. Colonies were amplified in 10 ml of LB medium containing ampicillin (100 ug/ml) and incubated at 37°C overnight under shaking (250 rpm). Aliquots were then used to inoculate larger volume cultures. Protein expression was induced until the OD600 (optical density at 600 nm) of the culture reached 0.6 by the addition of isopropyl-β-d-thiogalactopyranoside to a final concentration of 0.1 mM. His-tagged proteins were extracted by lysis and sonication, and extracts were clarified by centrifugation at 20,000g for 20 min. Fusion proteins were purified with Ni-NTA superflow beads (Qiagen). Purity of proteins was better than 80%, as determined by Coomassie Blue staining. HEK cell lysates expressing NBCe1-B and the kinases or phosphatases as indicated were incubated with Strep-tagged fusion proteins bound to the beads for 4 hours at 4°C. The beads were washed with lysis buffer, and proteins were released by incubation in SDS sample buffer at 56°C for 20 min and analyzed by SDS-PAGE and Western blotting.

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