The p3xFLAG-CMV-7.1/IRBIT, pCMV-HA-IRBIT, pEGFP-C1/NBCe1-B, pEGFP-C1/NBCe2-C, and pCDNA 3.1+/NBCe1-A constructs were described previously (28). pCDNA3-Flag CaN was a gift from R. Bassel-Duby and E. Olson (University of Texas Southwestern Medical Center), pCDNA-CaMKIIα was a gift from L. Nutt (St. Jude Children’s Research Hospital, Memphis), and pCDNA3/mPP1 was a gift from E. Delpire (Vanderbilt University Medical Center, Nashville). The cDNA encoding SPAK (a gift from M. Cobb, University of Texas Southwestern Medical Center) was excised from the original vectors and transferred to pCMV-myc. Point mutations were generated by site-directed mutagenesis with a Quick light chain mutagenesis kit (Agilent), using primers to change specific regions. All constructs were verified by sequencing of the entire open reading frames.

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