Species-specific knockdown of 190-kD ankyrin-G in rat myocytes using lentiviral shRNA. (A) Domain organization of 190-kD ankyrin-G with the site of shRNA target. Rat, mouse, and human shRNA nucleotide target sequences. Note that the human 190-kD ankyrin-G target sequence has three unique nucleotides in wobble base positions (asterisks) that render this target sequence resistant to the rat shRNA. (B) The 21-nucleotide target sequence (sense) is separated by a short loop spacer sequence followed by 21 nucleotides that form the reverse complement of the target sequence. (C) Rat and human species–specific shRNAs reduce 190-kD ankyrin-G expression. Rat myocytes or human HEK293 cells were transduced with control virus, rat ankyrin-G shRNA, or human ankyrin-G shRNA. Equal quantities of protein were analyzed by immunoblotting using affinity-purified ankyrin-G Ig or an antibody to an unrelated protein, NHERF1 (loading control). Note that the rat-specific ankyrin-G shRNA effectively reduces the expression of 190-kD ankyrin-G in rat cardiomyocytes. Moreover, the expression of 190-kD ankyrin-G is reduced in HEK293 cells transduced with human-specific ankyrin-G shRNA. (D) 190-kD ankyrin-G protein levels from whole cell lysates of rat cardiomyocytes transduced with control virus, human-specific ankyrin-G shRNA virus (hAnkG shRNA), or rat-specific ankyrin-G shRNA virus (rAnkG shRNA) were analyzed by immunoblotting and quantitated. Numbers represent the mean ± SD (error bars) from four independent experiments. *, P < 0.05.