Figure 4
GABA promotes GRP secretion mediated by GABBR1 in NE-like cells. (a) GABBR1 mRNA expression was evaluated by RT-qPCR of 96 h AR siRNA cells and 96 h control siRNA cells grown for 12 h in RPMI with indicated concentrations of baclofen. Differences among groups were compared by two-tailed unpaired Student’s t test, *p < 0.0001. (b) GABBR1 protein expression was evaluated by Western Blot in whole cell lysate. GAPDH was used as a loading control. Graphs represent the mean ± SEM (n = 4), *p < 0.001. The last result was normalized to 1 with respect to the vehicle. The concentrations of GRP in the medium of 96 h AR siRNA cells and 96 h control siRNA cells were examined by ELISA (n = 3/group). (c) GRP was present in the medium of both cells incubated with RPMI containing different concentrations of Baclofen (GABAB agonist) 1, 10 and 100 µM for 12 h. Differences among groups were compared by two-tailed unpaired Student’s t test, *p < 0.001 and **p < 0.0001. (d) GABAB antagonist (CGP 35348) was used, and cells were treated with 10, 50 and 100 µM in combination with baclofen (100 µm) for 12 h. Differences among groups were compared by two-tailed unpaired Student’s t test, *p < 0.001 and **p < 0.0001. The samples derive from the same experiment and that gels/blots were processed in parallel. Full-length blots/gels are presented in Supplementary Fig. 1.
