BK downregulates astrocytic marker GFAP expression in RBA cells. (A) Time dependence of BK-induced downregulation of GFAP protein expression. RBA cells were treated with 10 nM BK for the indicated times. The whole-cell lysates were analyzed by Western blot using an anti-GFAP or anti-GAPDH antibody. (B) The downregulation of GFAP by BK was confirmed by immunofluorescence in RBA cells. Cells were treated without (control) or with BK (10 nM) for 24 h and then labeled using an anti-GFAP antibody and a FITC-conjugated secondary antibody. Individual cells were imaged (scale bar = 20 μm) as described in ‘‘Methods’’. (C) Time dependence of BK-downregulated GFAP mRNA expression. RBA cells were treated with 10 nM BK for the indicated times. (D) Cells were pretreated with Hoe140 (Hoe, 10 μM), rottlerin (Rott, 1 μM), PD98059 (PD, 10 μM) for 1 h and then incubated with BK (10 nM) for 16 h. The total RNA was extracted and analyzed by RT-PCR as described under ‘‘Methods’’. Data are expressed as the mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01, as compared with control. ##
p < 0.01, as compared with BK alone. The image represents one of at least three individual experiments.