Fig. 2.

Induced expression of Na⁺/Ca²⁺ exchanger transgene does not change the expression of selected proteins involved in excitation-contraction coupling. Top: crude membranes were prepared from wild-type (WT), induced, and noninduced (non-Ind) mouse left ventricle (LV) and subjected to SDS-PAGE followed by Western blot analysis. Primary antibodies (see methods) were used to detect cardiac Na⁺/Ca²⁺ exchanger (NCX1), α₁ (alpha1)- and α₂ (alpha2)- subunits of Na⁺-K⁺-ATPase, sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2), unphosphorylated phospholemman (C2), phospholemman phosphorylated at serine68 (Cp68), cardiac ryanodine receptor (RyR2), high and low molecular weight phospholamban (PLB_H and PLB_L, respectively), and calsequestrin (CLSQ). PLB_H was detected under nonreducing conditions (only 1 band at ∼25 kDa), and PLB_L was detected under reducing conditions (2 major bands at ∼25 and ∼5 KDa; only the low molecular weight PLB is shown for PLB_L lanes). Composite results are shown in Table 2. Bottom: crude membranes prepared from rat and mouse LV were used to evaluate reactivity of polyclonal π11-13 antibody (1:500) against rat and mouse NCX1. To evaluate whether NCX1 density was similar between rat and mouse LV membranes, a monoclonal antibody (6H2; 1:1,000) raised against the NH₂ terminus of NCX1 was used.