NMDAR activation modulates NRG1-induced migration in ErbB4-transfected ST14A cells. A: ErbB4-transfected cells were cultured in 2% FBS DMEM for 18 hrs before total RNA isolation. Agarose gel electrophoresis was used to separate RT-PCR amplification products obtained with primer pairs specific for different NMDAR subunits (see Figure 1 for details). ErbB4-transfected cells express NR1.4a (arrows), NR2C, NR2D and NR3B (arrow). Note that NR3B was absent in wt ST14A cells. No PCR product was observed in reactions that omitted either reverse transcriptase (RT-) or starting material (C-, water). C- reaction contained all primer pairs. C+ = various brain regions from adult rats used as positive controls. B: As for wt ST14A cells, NMDA (1-10 μM) did not change ErbB4-transfected cell number. Cells were incubated in increasing concentrations of NMDA in 2% FBS DMEM for 18 hrs. Data are expressed as mean + SD (n = 3). C: Representative images of migrated cells showing that NMDA enhances NRG1-induced migration in ErbB4-transfected cells. Cells were treated either with 8 μM NMDA, 5 nM NRG1 or both for 18 hrs. Scale bar represents 200 μm. D: Histogram of the transwell experiment shown in C. Migration was calculated as percentage of total migrated cells as described in Material and Methods. Biological triplicate experiments were carried out in technical triplicate. Data are presented as means + SD. Statistical analysis was performed by applying two-way ANOVA, followed by Tukey's posthoc test (see text for details), ** = p < 0.01.