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Fig. 1. Protein expression in transgenic mouse lines. Approximately 10 mg of left ventricle tissue from transgenic myc-E22K, line 2 (L2) and line 4 (L4), myc-WT, line 1 (L1) and line 2 (L2) as well as Non-Tg mice were processed (see Materials and Methods) and quantitated on 15% SDS-PAGE using either Coomassie gel staining or polyclonal RLC CT-1 antibodies produced in this laboratory (raised against 15 residues from the C-terminus of human cardiac RLC). (A) SDS-PAGE stained with Coomassie and western blots of Non-Tg, Tg-WT (L1 and L2) and Tg-E22K (L2 and L4) expressed in mouse left ventricular tissue. Cardiac myofibrils prepared from left and right ventricular walls, septa and papillary muscles of transgenic mice were loaded at 10-20 µg per lane, run on 15% SDS-PAGE for Coomassie staining, while approximately 0.1-10 µg of the left-ventricular (LV) muscle extracts were loaded per lane for western blotting. The blots were calibrated with purified recombinant myc-WT that showed a linear dependence in the range of 10-75 ng. The highest loading concentration of 75 ng is shown in the right lane of each blot. The endogenous RLC and transgenic myc-WT, and myc-E22K proteins were all quantitated using polyclonal RLC CT-1 antibodies. Note that transgenic myc-WT and myc-E22K proteins migrate slower than the endogenous RLC because of the myc sequence attached to their N-termini. This was true for the Coomassie-stained gel (left upper panel) and also for RLC-antibody-stained western blots (right panels). Alternatively, the amount of transgene expression was quantitated using monoclonal myc-antibodies (clone 9E10) raised to a peptide from the human MYC protein (left lower panel). (B) Visualization of western blots with fluorescence. Western blots treated with polyclonal RLC CT-1 antibodies were further processed with fluorescent secondary antibodies conjugated with fluorescent dye Cy 5.5. Like in Fig. 1A, myc-RLC migrates slower than endogenous RLC because of the additional myc sequence attached to the N-terminal region of Tg-E22K or Tg-WT proteins. Note that no background fluorescence was found in the blot, indicating that no nonspecific antibody binding or ectopic transgene expression occurred. (C) Multi-determination of transgenic protein expression. Coomassie-stained gels as well as standard and fluorescent western blots were quantitated. Percentages of protein expression were determined by using Coomassie staining (black bars), western blots `standard' (gray bars) and western blots `fluorescence' (striped bars) were as follows: Tg-WT L1, 8.9±5.0 (n=12), 11.9±5.2 (n=6) and 7.4±2.6 (n=8); Tg-WT L2, 24.5±3.8 (n=12), 29.2±8.8 (n=3) and 23.1±3.1 (n=9); Tg-E22K L2, 69.8±2.9 (n=12), 60.4±6.0 (n=6) and 65.4±7.1 (n=8) and Tg-E22K L4, 86.8±4.3 (n=12), 92.3±2.9 (n=12) and 80.7±3.0 (n=4), respectively. Data are expressed as the average of n measurements ± s.d.
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