FIG. 9.

ErbB2 expression knockdown by targeted siRNAs attenuates neuregulin-1β (NRG)-activated AKT and Erk1/2 phosphorylation. HiPSC-CMs were incubated with Lipofectamine/Opti-MEM (transfection vehicle), 100-nM scrambled or targeted siRNAs [control (ctrl) or ErB2 siRNAs] for up to 144 h. To test the effect of ErbB2 siRNA on NRG-activated AKT or Erk1/2 phosphorylation, cells at 72-h transfection were exposed to 100-ng/ml NRG for 30 min prior to lysing in RIPA buffer. (A) and (B) Representative Western blots of three independent experiments show diminished ErB2 expression and reduced phosphorylation of AKT and Erk1/2 in response to 100-ng/ml NRG treatment. Thirty microgram total protein was loaded into each individual well of a 10-well mini-gel (30 μl/well) (A); 10 μg total protein was loaded into each individual well of a 10-well mini-gel (30 μl/well) (B). Blots were first probed for phosphorylated AKT(Ser473) and Erk1/2(Thr202/Tyr204), then stripped and re-probed for total AKT and Erk1/2, and GAPDH. (C) Quantification of ErbB2 expression after 72- or 144-h transfection. ErbB2 expression relative to the protein loading (GAPDH) was normalized to the transfection vehicle group. (D) and (E) Quantification of NRG-induced AKT and Erk1/2 phosphorylation at 72-h transfection. Proteins detected by anti-pAKT(Ser473) or anti-pErk/12(Thr202/Tyr204) were expressed as a fraction of that detected by anti-total AKT or Erk1/2 antibodies. Each data point represents the mean ± SE of three independent experiments. *p < 0.05 compared with control siRNAs, ^‡p < 0.05 compared with 72-h ErbB2 siRNAs, ^#p < 0.05 compared with vehicle control exposed to NRG.