Pdsred monomer c1

The α1A‐GFP (green fluorescent protein) and α1A‐NLSmut constructs were described previously.^{8 (link),16 (link)} PKCδ constructs were made using a human cDNA encoding PKCδ (GenBank L07860), amplified by PCR with primers (IDT) containing Xho I (5′) and Kpn I (3′) restriction sites, and subcloned into the multicloning site of pDsRed monomer‐C1 (Clontech Laboratories, Mountain View, CA) in frame. Dominant negative PKCδ (PKCδ‐DN) was made by a K→A mutation at position 378 by site‐directed mutagenesis using the QuikChange kit (Stratagene, La Jolla, CA).^{20 (link)} The PKCδ nuclear localization mutant (PKCδ‐NLSmut) was made with a DNA minigene (IDT) containing the PKCδ NLS flanked by unique XcmI and AscI sites, but with the arginines (R) or lysines (K) replaced with alanine (A) at positions 613, 614, 615, 621, 623, 625, and 628 corresponding to the human sequence.^{20 (link)} All DsRed‐PKCδ constructs were subcloned into the AdEasy system (Stratagene) for adenovirus production. For all experiments, cardiac myocytes were infected at a multiplicity of infection of 1000 for the α1A‐AR constructs, resulting in 2.5‐fold overexpression for the α1A‐AR,^{8 (link)} and 5‐fold overexpression for the PKCδ constructs.

Open reading frames of Cdc6 derivatives were cloned into the vector pDsRed-Monomer-C1 (Clontech), unless indicated otherwise. DNA constructs were transfected into cells using Lipofectamine 2000 transfection reagent (Invitrogen). The following siRNA oligonucleotides were purchased from Samchully Pharmaceutical: control GL3 siRNA, 5′-CUUACGC UGAGUACUUCGATT-3′, and Cdc6 siRNA, 5′-UAAGCCGGA UUCUGCAAGA-3′ (Lee et al., 2017 (link)). These siRNA oligonucleotides were transfected into cells using Oligofectamine (Invitrogen).