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Figure 4


Fig. 4. Effects of knockdown of centaurin {alpha}1 protein by siRNA in dissociated cultured hippocampal neurons. Hippocampal neurons were transfected after dissociation (0 DIV) and cultured for 3, 7 and 14 DIV. (A,B) Immunoblot analysis of endogenous centaurin {alpha}1 in hippocampal cultures. Total cell lysates (12.5 µg lysate per lane) were probed with anti-centaurin {alpha}1 and an anti-actin antibodies, which were used to assess actin levels for calculation of the knockdown percentage. (A) Lane 1, scrambled (Scr) siRNA 3 DIV; lane 2, rat centaurin {alpha}1 (rCena1) siRNA 3 DIV; lane 3, Scr siRNA 7 DIV; lane 4, rCena1 siRNA 7 DIV; lane 5, Scr siRNA 14 DIV; lane 6, rCena1 siRNA 14 DIV. (B) Lane 1, Scr siRNA; lane 2, rCena1 siRNA; lane 3, rCena1 siRNA plus human Flag-centaurin {alpha}1. (C) Indirect immunofluorescence showing endogenous centaurin {alpha}1 (red) compared with beta-tubulin (green) to visualize neuronal morphology at 3 and 7 DIV. Double-stranded scrambled RNA control is shown in left-hand panels and double-stranded siRNA to rat centaurin {alpha}1 in the right-hand panels. Bars, 20 µm. (D) Rescue by expression of human centaurin {alpha}1. Neurons were transfected with rat centaurin {alpha}1 siRNA, GFP and human centaurin {alpha}1. Indirect immunofluorescence showing endogenous plus heterologous centaurin {alpha}1-(red), beta-tubulin (blue) to visualize neuronal morphology and GFP (green) to label neurons co-expressing human Flag centaurin {alpha}1 at 3 and 7 DIV. Bars, 20 µm. (E) Quantification of the effects of siRNA knockdown of centaurin {alpha}1 at 3 and 7 DIV and rescue with human centaurin {alpha}1 compared with scrambled siRNA control. The data were quantified from three independent experiments (n=30 at 3 DIV, n=25 at 7 DIV cells for each condition). Data represent mean ± s.e. *P<0.05.