Figure 2. siRNA directed knock-down of AKAP150 expression in TG neurons.

A.) Cultured TG neurons transfected with nothing (−), mock transfected (Mock), scrambled/control negative silencer #2 siRNA (Scram), AKAP150-1 siRNA (AKAP-1), or AKAP150-2 siRNA (AKAP-2). Western blot expression of AKAP150 (above) following transfection quantified and normalized to expression of TRPV1.

B.) Cultured TG neurons transfected with AKAP150-1 harvested day 0, 1, 2, 3 or 4 following transfection. Western blot expression of AKAP150 (above) following transfection quantified and normalized to expression of TRPV1.

C.) Real-time RT-PCR experiments were performed with RNA samples from cultured TG neurons left alone (control), mock transfected (Mock), transfected with scrambled/control negative silencer #2 control siRNA (Scram), or with AKAP150-1 (AKAP-1 siRNA) following 1 day post-transfection. Reactions were performed using primers specific for AKAP150 (PRKA) gene and the internal control (18S). Data were normalized to the relative amount of control AKAP150 mRNA/18S. Data are presented as mean ± SEM (n = 6 per group).

D.) Cultured TG neurons mock transfected (Mock), with scrambled/control negative silencer #2 control siRNA (Scram), or with AKAP150-1 (AKAP-1) were analyzed for ³²P-incorporation following vehicle (H₂0) or 8-Br-cAMP (10 μM, 5 min). Autoradiographic results of labeled TRPV1 quantified and normalized to total TRPV1 expression.

E.) Cultured TG neurons mock transfected or with AKAP150-1 siRNA were surface biotinylated and analyzed for TRPV1 plasma membrane expression relative to total cell expression of AKAP150.

All plotted data are expressed as mean ± SE, **p<0.01, ***p<0.005, NS = not significant.