Figure 1.

Ca_V1.2 transgene injection construct and expression analysis. (A) Schematic of the injection construct consisting of the CAG promoter, Loxp-3xStop-Loxp cassette cassette, 5′ UTR, Ca_V1.2^Tg and 3′ UTR, as well as PCR primer and poly-A consensus sequence locations. The 12-kB transgene was linearized for injection using NotI restriction enzyme digestion. (B) Cartoon of the Ca_V1.2^Tg protein product including the location of the HA epitope. (C) The Ca_V1.2^Tg injection construct was expressed in HEK293 alone or with pCAG-ERT2-Cre-ERT2. Blots containing whole-cell lysates were probed with rat anti-HA to detect the presence of the transgenic protein and anti β-actin as a loading control. Only those samples co-expressing Cre recombinase (Ca_V1.2^Tg+; Syn^Tg+) also expressed Ca_V1.2^Tg. (D) Representative Western blots containing cell lysates from whole-brain tissue. Membranes were probed with either anti-Ca_V1.2 or anti-HA where appropriate and anti β-actin as a loading control. (D₁) Immunoblotting with an HA-specific antibody reveals staining that is only present in tissue harvested from Ca_V1.2^Tg+; Syn^Tg+ mice. (D₂) Western blot of total Ca_V1.2 protein (transgenic and endogenous). (E) Quantification of total Ca_V1.2. Ca_V1.2^Tg+; Syn^Tg+ mice express approximately 100% more Ca_V1.2 than their control or Ca_V1.2^Tg+; Syn^Tg− littermates (F_2,18 = 1.612, p = .0013; significant post hoc comparisons: wild-type × Ca_V1.2^Tg+; Syn^Tg+, ∗∗p = .007. Ca_V1.2^Tg+; Syn^Tg− × Ca_V1.2^Tg+; Syn^Tg+, ∗∗p = .0018). All data are presented as mean ± SEM. HA, hemagglutinin; PCR, polymerase chain reaction; UTR, untranslated region.