Fig. 3.

Calcineurin/NFAT is required for NRG1 and ErbB signaling. (A) Ca²⁺ influx stimulated by NRG1. DRG cocultures were preloaded with the Ca²⁺ sensor Fura-2AM in the presence or absence of PLC-γ inhibitor U73122, 30 min before stimulation. Ca²⁺ influx was detected, and average Ca²⁺ levels were measured. (B) Dephosphorylation of NFATc3 and c4 is not induced in mutant cocultures after NRG1 stimulation, as examined by immunoblotting (ib). Arrows, dephosphorylated (active) NFATc3 and c4. (C) Tyrosine phosphorylation levels of ErbB3 following the activation by NRG1. ErbB3 were isolated by immunoprecipitation (ip). Tyrosine phosphorylation was detected by immunoblotting using phosphotyrosine-specific antibody. The blot was reprobed with ErbB3-specific antibody to verify protein expression levels. (D) Calcineurin/NFAT-dependent transcription is stimulated by NRG1. E13.5 DRG cocultures were transfected with luciferase reporter constructs containing the DSCR1 enhancer, NFATc4 promoter, Krox20 promoter, or Krox20 MSE. Luciferase activities were measured after 20 hours of NRG1 stimulation. Fold activation is relative to unstimulated cells (n = 6; error bars, +SEM; **P < 0.01; ***P <0.001; t test).