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. 2013 Aug 31;288(41):29403–29413. doi: 10.1074/jbc.M113.470708

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — Cypher/ZASP tethered PKA to phosphorylate the LTCC cytosolic region at Ser¹⁹²⁸in vitro. A, the Cypher PDZ domain interacted with the LTCC C-terminal PDZ binding motif (VSSL). Myc-tagged Cypher or its PDZ domain deletion mutant (ΔPDZ) was co-expressed with FLAG-tagged LTCC cytosolic region (AA1510–2172) or its deletion mutant (ΔVSSL) in HEK293 cells. FLAG-tagged proteins were enriched by anti-FLAG antibody, and interacting proteins were verified by blotting with an anti-Myc antibody. IP, immunoprecipitation; IB, immunoblotting. B, FLAG-tagged LTCC cytosolic region with or without Myc-tagged Cypher was co-expressed in HEK293 cells. Forskolin (100 μm, 30 min) was used to activate PKA. LTCC protein was purified by anti-FLAG antibody, and the phosphorylation was detected by a phospho-(Ser/Thr) PKA substrate antibody. IBMX, 3-isobutyl-1-methylxanthine. C, LTCC and its mutants S1928A and C-terminal VSSL deletion (ΔVSSL) were expressed in HEK293 cells with Cypher. LTCC phosphorylation with or without the PKA agonist forskolin treatment was assessed.