The neuronal Arf GAP centaurin 1 modulates dendritic differentiation

JCS006346 Supplementary Material

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• Supplemental Figure S1 -

  Fig. S1. Developmental expression of endogenous centaurin α1 in dissociated primary cultured hippocampal neurons. (A) Indirect immunofluorescence was used to visualize endogenous centaurin α1 expression pattern at 3, 7, 14, 21 DIV. Bar, 10 μm.

• Supplemental Figure S2 -

  Fig. S2. Localization of endogenous centaurin α1 in dissociated primary cultured hippocampal neurons. Indirect immunofluorescence was used to visualize endogenous centaurin α1 localization (red) compared with SV2 and β-tubulin (B-tubulin). Arrows denote regions of juxtaposition. Bars, 5 μm. Quantification of the percentage colocalization of centaurin α1 with the neuronal markers: microtubule associated protein 2 (MAP-2), tau, synaptophysin (syn), SV2, PSD-95 and spinophilin.

• Supplemental Figure S3 -

  Fig. S3. Effects of knockdown of centaurin α1 protein by siRNA in dissociated primary cultured hippocampal neurons. Hippocampal neurons were transfected with double-stranded RNA by Amaxa transfection after dissociation (0 DIV) and cultured for 3, 7 or 14 DIV. (A) Indirect immunofluorescence showing endogenous centaurin α1 (red) compared with β-tubulin (green) to visualize neuronal morphology at 3 and 7 DIV. By 14 DIV, the neuronal morphology has recovered as assessed by fluorescence of GFP-transfected neurons (green), which allows better visualization of filopodia and lamellipodia. Double-stranded scrambled RNA control (left panels), double-stranded siRNA to centaurin α1 (right panels). Bars, 20 μm. (B) Rescue by expression of human centaurin α1. Neurons were transfected with rat centaurin α1 siRNA, GFP and human centaurin α1. Indirect immunofluorescence showing endogenous plus heterologous centaurin α1 (red), β-tubulin (blue) to visualize neuronal morphology and GFP (green) to mark neurons co-expressing human Flag centaurin α1 at 3 and 7 DIV. Bars, 20 μm.

• Supplemental Figure S4 -

  Fig. S4. Effects of overexpression of wild-type centaurin α1 in dissociated hippocampal neurons in early development. Hippocampal neurons were transfected by nucleofection after dissociation, and cultured for 3, 7 and 14 days. (A) Immunoblot analysis of endogenous centaurin α1 in hippocampal neurons. Total lysates (12.5 μg protein per lane) on blots were probed with anti-centaurin α1 antibody and an anti-actin antibody were used as a loading control to assess actin levels to calculate the fold overexpression of centaurin α1. (B) Fluorescence (GFP-green) and indirect immunofluorescence of β-tubulin blue (7 & 14 div), 7 and 14 DIV Flag-tagged centaurin α1 (red) were used to visualize transfected neurons and morphological effects. Expression of GFP control (left panels) compared with GFP plus Flag-centaurin α1 (right panels). Bars, 20 μm. Boxed areas show a higher magnification

• Supplemental Figure S5 -

  Fig. S5. Effects of overexpression of a GAP inactive mutant centaurin α1 on dendritic branching and outgrowth. Hippocampal neurons were transfected and cultured for 7-14 days. Fluorescence (GFP-green) and indirect immunofluorescence (β-tubulin-red) were used to visualize transfected neurons and morphological effects. Expression of GFP control (left panels) compared with GFP plus Flag-R49Kcentaurin α1 (right panel). Bars, 20 μm. Boxed areas show a higher magnification.