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

FIGURE 3.

FIGURE 3.

Effect of the T76S/T81A mutation of Rab35 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 shRab35 together with pEGFP-C1 (top row) or together with pEGFP-C1-Rab35SR(WT, T76S/T81A, or S5A) (bottom row). The cells were fixed after NGF stimulation for 36 h and examined under a confocal microscope. Note that knockdown of Rab35 in PC12 cells inhibited neurite outgrowth (top right panel), whereas re-expression of Rab35SR(WT) (bottom left panel), but not of Rab35SR(T76S/T81A) (bottom middle panel) or Rab35SR(S5A) (bottom right panel), in shRab35-treated PC12 cells restored neurite outgrowth. Under our experimental conditions, manipulation of Rab35 had no significant effect on the number of neurites (shControl, 2.2 ± 0.06; shRab35, 2.0 ± 0.04; shRab35 + Rab35SR(WT), 2.3 ± 0.06; shRab35 + Rab35SR(T76S/T81A), 2.1 ± 0.05; and shRab35 + Rab35SR(S5A), 1.9 ± 0.05 (mean ± S.E.)), suggesting that Rab35 is involved in neurite extension rather than in neuritogenesis. 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 < 0.01; ***, p < 0.001 (one-way analysis of variance followed by the Tukey-Kramer test). C, equivalent expression level of EGFP-Rab35SR(WT, T76S/T81A, or S5A) in shRab35-expressing PC12 cells. Cell lysates of PC12 cells expressing shRab35 together with EGFP-Rab35SR(WT, T76S/T81A, or S5A) were subjected to 12.5% SDS-PAGE followed by immunoblotting with anti-Rab35 antibody (top panel; 1:1,000 dilution) and anti-actin antibody (bottom panel; 1:20,000 dilution). The positions of the molecular mass markers (in kilodaltons) are shown on the left.