Calmodulin phosphorylation and modulation of endothelial nitric oxide synthase catalysis -- Data Supplement - HTML Page - 06377SuppMethods.htslp -- Proceedings of the National Academy of Sciences

Supporting Methods
Cloning of the CaM Plasmid Constructs.
The plasmid consisting of the CaM cDNA in the pMEX8 vector was the template for PCRs that generated fragments used for subcloning CaM WT and CaM S81A, S81D, and T79D/S81D mutant cDNAs in the pET23(+) prokaryotic expression vector (Novagen). For the WT CaM construct, the primers used were the BamH1 forward primer (5’-CGGGATCCAAGAAGGATACATAATGGCTGACCAACTCACCG-3’) and the Sal1 reverse primer (5’-CGATAGCGTCGACTTCTAGAGTCACTTAGCCGTCATC-3’). Two-step overlap extension PCR was used for generating the S81A, S81D, and T79D/S81D constructs. In the first step, the two primer pairs were the following: (i) the BamH1 forward primer and either the S81A reverse primer (5’-GATCTCCTCTTCGGCGTCCGTGTCCTTC-3’), the S81D reverse primer (5’-GATCTCCTCTTCGTCGTCCGTGTCCTTC-3’), or the T79D/S81D reverse primer (5’-CCTCTTCGTCGTCATCGTCCTTCATCTTCC-3’); and (ii) either the S81A forward primer (5’-GAAGGACACGGACGCCGAAGAGGAGATC-3’), the S81D forward primer (5’-GAAGGACACGGACGACGAAGAGGAGATC-3’) or the T79D/S81D forward primer (5’-GGAAGATGAAGGACGATGACGACGAAGAGG-3’) and the Sal1 reverse primer. In the second step, the BamH1 forward primer and the Sal1 reverse primer were used to amplify the whole fragment. The full-length PCR fragments were subcloned into the pET-23(+) vector between the BamH1 and Sal1 sites.

The pET23(+) vectors encoding the CaM WT or S81A plasmids served as templates for generating the FLAG epitope-tagged CaM WT and S81A constructs, respectively. The primers used were the BamH1 forward primer and the FLAG.Xho1 reverse primer (5’-CCAGTCTCGAGTCACTTATCATCGTCGTCCTTGTAATCCTTAGCCGTCATCATTTGC-3’). The PCR products were subcloned into the eukaryotic expression vector pcDNA3 (Invitrogen) between the BamH1 and Xho1 sites. In addition, the FLAG epitope-tagged CaM S81D mutant was generated by subcloning the pET23(+) S81D CaM construct into the FLAG-tagged pcDNA3 S81A CaM at the internal EcoR1 and Cla1 sites.

The plasmid encoding the FLAG-tagged CaM T79A/S81D in pcDNA3 was generated by using the FLAG-tagged CaM S81D in pcDNA3 as the template for a two-step overlap extension PCR. In the first step, the two primer pairs were: (i) the BamH1 forward primer and the T79A/S81D reverse primer

(5’-CCTCTTCGTCGTCCGCGTCCTTCATCTTCC-3’); and (ii) the T79A/S81D forward primer (5’-GGAAGATGAAGGACGCGGACGACGAAGAGG-3’) and the FLAG.Xho1 reverse primer. In the second step, the BamH1 forward primer and the FLAG.Xho1 reverse primers were used to amplify the whole fragment.

The FLAG epitope-tagged CaM S101D and CaM T79D/S81D/S101D constructs were generated in two steps. First, PCRs were performed with the pET23(+) WT CaM or T79D/S81D CaM construct used as the template and the BamH1 forward primer and the 101D.Pml1 reverse primer (5’-CGTCATCACGTGACGTAGCTCAGCAGCGTCGATGTAGCCGTTGCCATCC-3’). The PCR products were then subcloned into the construct encoding the FLAG epitope-tagged WT CaM in pcDNA3 between the BamH1 site and the internal Pml1 site.