Journal: Molecular Biology of the Cell

Article Title: Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

doi: 10.1091/mbc.e11-03-0277

Figure Lengend Snippet: FIGURE 3: NGF-induced endocytosis of pTrkA into Rab22-containing early endosomes. (A) Confocal fluorescence microscopy showing the localization of pTrkA and RFP-Rab22 in PC12 cells, in the absence of NGF. Cells overexpressing TrkA and RFP-Rab22 were immunostained with the anti-pTrkA polyclonal antibody and goat anti–rabbit IgG conjugated with Alexa Fluor 488. Scale bar: 12 μm. (B) Confocal fluorescence microscopy showing colocalization of pTrkA with RFP-Rab22 on large early endosomes in PC12 cells, upon NGF treatment. Cells overexpressing TrkA and RFP-Rab22 (either WT or S19N, as indicated) were treated with NGF (100 ng/ml) for 30 min and then immunostained with the anti-pTrkA polyclonal antibody and goat anti–rabbit IgG conjugated with Alexa Fluor 488, followed by confocal fluorescence microscopy. Scale bar: 12 μm. (C) Confocal fluorescence microscopy showing NGF-induced endocytosis of pTrkA into large endosomes in PC12 cells. Cells overexpressing TrkA were treated with NGF for different times as indicated, and then immunostained with the anti-pTrkA polyclonal antibody and goat anti–rabbit IgG conjugated with Alexa Fluor 488, as described above. Scale bar: 12 μm.

Article Snippet: Monoclonal antibodies for actin and Myc were purchased from Sigma-Aldrich (St. Louis, MO), whereas polyclonal antibodies for Rab22 and phospho-TrkA (at Tyr490) were from ProteinTech Group (Chicago, IL) and Cell Signaling Technology (Beverly, MA), respectively.

Techniques: Fluorescence, Microscopy

Journal: Molecular Biology of the Cell

Article Title: Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

doi: 10.1091/mbc.e11-03-0277

Figure Lengend Snippet: FIGURE 4: Inhibitory effect of Rab22 knockdown on the endocytosis and localization of pTrkA on endosomes. (A) Confocal fluorescence microscopy showing pTrkA endocytosis in PC12 cells expressing the Rab22 shRNA (top panels) or the scrambled shRNA (bottom panels), in response to NGF treatment (100 ng/ml) for the indicated times. The vector expressing the shRNAs also expressed DsRed, and the cells were immunostained with the anti-pTrkA polyclonal antibody and goat anti–rabbit IgG conjugated to Alexa Fluor 488. Scale bar: 12 μm. (B) Quantification of the effect of Rab22 shRNA on pTrkA endocytosis into intracellular endosomes. Fifty cells expressing the scrambled shRNA or the Rab22 shRNA described in (A) were counted to determine the percentage of cells containing pTrkA-positive endosomes. Two-way ANOVA was performed (***p < 0.001); error bars indicate SEM of triplicate samples.

Article Snippet: Monoclonal antibodies for actin and Myc were purchased from Sigma-Aldrich (St. Louis, MO), whereas polyclonal antibodies for Rab22 and phospho-TrkA (at Tyr490) were from ProteinTech Group (Chicago, IL) and Cell Signaling Technology (Beverly, MA), respectively.

Techniques: Knockdown, Fluorescence, Microscopy, Expressing, shRNA, Plasmid Preparation