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. 2015 Feb 18;290(14):9064–9074. doi: 10.1074/jbc.M114.611301

FIGURE 7.

FIGURE 7.

Effect of the N610A/N691A mutation of centaurin-β2 on NGF-induced neurite outgrowth of PC12 cells. A, typical images of PC12 cells (merged bright field images and EGFP fluorescence images) transiently expressing shControl or shCentβ2 together with pEGFP-C1 (top row) or pEGFP-C1-Centβ2SR(WT, N610A/N691A, or ΔANKR) (bottom row). The cells were fixed after NGF stimulation for 36 h and examined under a confocal microscope. Note that knockdown of centaurin-β2 in PC12 cells inhibited neurite outgrowth (top right panel), whereas re-expression of Centβ2SR(WT) (bottom left panel), but not of Centβ2SR(N610A/N691A) (bottom middle panel) or Centβ2SR(ΔANKR) (bottom right panel), in shCentβ2-treated PC12 cells restored total neurite length. Under our experimental conditions, manipulation of centaurin-β2 had no significant effect on the number of neurites (shControl, 2.2 ± 0.05; shCentβ2, 2.0 ± 0.04; shCentβ2 + Centβ2SR(WT), 2.1 ± 0.04; shCentβ2 + Centβ2SR(N610A/N691A), 2.0 ± 0.04; and shCentβ2 + Centβ2SR(ΔANKR), 2.0 ± 0.05 (mean ± S.E.)), suggesting that centaurin-β2 is involved in neurite extension rather than in neuritogenesis, the same as Rab35 is. Scale bars, 20 μm. B, quantification of total neurite length shown in A (sum of the lengths of the broken white lines in each PC12 cell). The bars represent the means and S.E. of data from three independent experiments (n = 100 cells; more than 30 cells were analyzed in each experiment). ***, p < 0.001 (one-way analysis of variance followed by the Tukey-Kramer test). C, equivalent expression level of EGFP-Centβ2SR(WT, N610A/N691A, or ΔANKR) in shCentβ2-expressing PC12 cells. Cell lysates of PC12 cells expressing shCentβ2 together with EGFP-Centβ2SR(WT, N610A/N691A, or ΔANKR) were subjected to 10% SDS-PAGE followed by immunoblotting with anti-Centβ2 antibody (top panel; 1:500 dilution), anti-GFP antibody (middle panel; 1:1,000 dilution), and anti-actin antibody (bottom panel; 1:20,000 dilution). Because our anti-Centβ2 antibody recognized the C-terminal domain of centaurin-β2, it failed to detect EGFP-Centβ2SR(ΔANKR) expression (top panel, lane 5). The positions of the molecular mass markers (in kilodaltons) are shown on the left.