cd 107a  (Miltenyi Biotec)

Journal: The Journal of Neuroscience

Article Title: Rho Regulates Membrane Transport in the Endocytic Pathway to Control Plasma Membrane Specialization in Oligodendroglial Cells

doi: 10.1523/JNEUROSCI.4926-06.2007

Figure Lengend Snippet: Subcellular localization of PLP is regulated by Rho GTPases and tyrosine kinases. Oli-neu cells stably expressing PLP-EGFP were treated for 16 h with Genistein, Y27632, C3 transferase, or conditioned medium from primary cultures of astrocytes. A, The subcellular localization of PLP (green) was determined by staining living cells at 4°C with the O10 antibody to detect PLP on the cell surface (red) or by staining for Lamp-1 (red). Quantitative analysis of colocalization of PLP-EGFP with O10 (B) and Lamp-1 (C) is shown. Values are given as the mean ± SE (n > 40 cells; *p < 0.05; t test). Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used: hemagglutinin (HA) (16B12, mouse monoclonal; Covance, Berkley, CA), HA (rabbit polyclonal IgG; Abcam, Cambridge, UK), GFP (polyclonal rabbit; Abcam), GM130 (mouse monoclonal IgG1; BD Biosciences, Franklin Lakes, NJ), Lamp-1 (CD 107a, rat monoclonal; BD Biosciences), myc (rabbit polyclonal IgG; Upstate Biotechnology, Temecula, CA), myc (monoclonal IgG; Cell Signaling Technology, Denver, CO), O10 (monoclonal mouse IgM) ( Jung et al., 1996 ), RhoA (26C4, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), and Src (36D10, rabbit monoclonal IgG; Cell Signaling Technology).

Techniques: Stable Transfection, Expressing, Staining

Journal: The Journal of Neuroscience

Article Title: Rho Regulates Membrane Transport in the Endocytic Pathway to Control Plasma Membrane Specialization in Oligodendroglial Cells

doi: 10.1523/JNEUROSCI.4926-06.2007

Figure Lengend Snippet: Neurons reduce the endocytosis of GPI-CFP to LE/Ls in Oli-neu cells. A, Cells were treated with C3 transferase, Genistein, or conditioned neuronal medium (cnm) as indicated above (Fig. 3), and the uptake of GPI-CFP was analyzed by antibody uptake experiments. Cells were labeled at 4°C with a polyclonal antibody against GFP and then warmed to 37°C for 30 min to allow internalization of antibodies bound to GPI-CFP. Fixed, nonpermeabilized cells were labeled with Cy5-conjugated secondary antibody to visualize GPI-CFP at the cell surface (blue) and then permeabilized and labeled with Cy3-conjugated secondary antibodies to resolve internalized protein (red). By image analysis, we determined the ratio of Cy3- to Cy5-labeled GPI-CFP to obtain a value for the amount of protein internalized. Values represent the mean ± SE (n > 30 cells; **p < 0.01; ***p < 0.001; t test). B, Oli-neu cells were transfected to express GPI-EGFP, incubated with conditioned neuronal medium (cnm) for 16 h or not incubated (ctrl), and treated with cycloheximid for the last 4 h of the incubation period to inhibit protein synthesis and to chase GPI-EGFP from the biosynthetic pathway. Cells were processed and analyzed by immunofluorescence for Lamp-1 (red) and GPI-EGFP (green) colocalization. C, The endocytosis of GPI-CFP was followed by antibody-internalization experiments (for 45 min at 37°C). Cells were labeled at 4°C with polyclonal antibodies against GFP (anti-GFP) and then warmed to 37°C to allow internalization of antibodies bound to GPI-CFP together with Tf. Cells were fixed, permeabilized, and labeled with Alexa 488-conjugated secondary antibodies to resolve internalized GPI-CFP. Note the colocalization of anti-GFP (green) with Lamp-1 (blue) but not with Tf. Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used: hemagglutinin (HA) (16B12, mouse monoclonal; Covance, Berkley, CA), HA (rabbit polyclonal IgG; Abcam, Cambridge, UK), GFP (polyclonal rabbit; Abcam), GM130 (mouse monoclonal IgG1; BD Biosciences, Franklin Lakes, NJ), Lamp-1 (CD 107a, rat monoclonal; BD Biosciences), myc (rabbit polyclonal IgG; Upstate Biotechnology, Temecula, CA), myc (monoclonal IgG; Cell Signaling Technology, Denver, CO), O10 (monoclonal mouse IgM) ( Jung et al., 1996 ), RhoA (26C4, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), and Src (36D10, rabbit monoclonal IgG; Cell Signaling Technology).

Techniques: Labeling, Transfection, Incubation, Immunofluorescence

Journal: The Journal of Neuroscience

Article Title: Rho Regulates Membrane Transport in the Endocytic Pathway to Control Plasma Membrane Specialization in Oligodendroglial Cells

doi: 10.1523/JNEUROSCI.4926-06.2007

Figure Lengend Snippet: Regulation of LE/L dynamics by RhoB and tyrosine kinase activity. A, Living Oli-neu cells expressing PLP-EGFP (green) were treated with C3 transferase or Genistein, labeled with LysoTracker (red), and observed by confocal microscopy. Images were collected every ∼15 s. A representative example of a time stack is shown. The images at the right of each time stack show a merged image (see Material and Methods) with the first image in red and movement over a ∼130 s period in green. Relative motility of LE/Ls was quantified by determining the ratio of the integrated intensity of green to red pixels (see Material and Methods). Examples of moving vesicles are highlighted with arrows. Values represent the mean ± SE (n > 20 cells; **p < 0.01; t test). B, C, Oli-neu cells (B) and primary cultures of oligodendrocytes (C) (for 2 d in culture) were pretreated with 10 μm Y27632 for 2 h or left untreated, and the motility of LysoTracker-labeled vesicles was determined as in A. Primary oligodendrocytes were identified by incubating living cells with O4 and Alexa 488-conjugated secondary antibody. Values represent the mean ± SE (n > 20 cells; *p < 0.05; **p < 0.01; t test). D, Oli-neu cells were cotransfected to express PLP-EGFP and HA-tagged wild-type (wt), constitutively active (ca), and constitutively inactive (dn) versions of RhoB. Colocalization with PLP (green) and Lamp-1 (red) was observed for wild-type and the constitutively active versions of RhoB (blue). E, Oli-neu cells were transfected to express membrane-targeted GFP (ctrl) or the dominant-negative version of RhoB (T19N) fused to GFP (RhoB dn), labeled with LysoTracker red, and imaged by confocal microscopy. Changes in mobility were determined as described in A. Values represent the mean ± SE (n > 20 cells; *p < 0.05; t test). F, Oli-neu cells were transfected to express membrane-targeted GFP (ctrl) or the constitutively active versions of RhoB, RhoA, and RhoC fused to GFP treated with conditioned neuronal medium (cnm) for 12 h and labeled with LysoTracker red, and changes in mobility were determined as described in A. Values represent the mean ± SE (n > 20 cells; **p < 0.01; ***p < 0.001; t test). Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used: hemagglutinin (HA) (16B12, mouse monoclonal; Covance, Berkley, CA), HA (rabbit polyclonal IgG; Abcam, Cambridge, UK), GFP (polyclonal rabbit; Abcam), GM130 (mouse monoclonal IgG1; BD Biosciences, Franklin Lakes, NJ), Lamp-1 (CD 107a, rat monoclonal; BD Biosciences), myc (rabbit polyclonal IgG; Upstate Biotechnology, Temecula, CA), myc (monoclonal IgG; Cell Signaling Technology, Denver, CO), O10 (monoclonal mouse IgM) ( Jung et al., 1996 ), RhoA (26C4, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), and Src (36D10, rabbit monoclonal IgG; Cell Signaling Technology).

Techniques: Activity Assay, Expressing, Labeling, Confocal Microscopy, Transfection, Dominant Negative Mutation

Journal: The Journal of Neuroscience

Article Title: Rho Regulates Membrane Transport in the Endocytic Pathway to Control Plasma Membrane Specialization in Oligodendroglial Cells

doi: 10.1523/JNEUROSCI.4926-06.2007

Figure Lengend Snippet: Changes in c-Src activity regulate the dynamics of LE/L. A, Oli-neu cells were incubated with conditioned neuronal medium (cnm) for 16 h or left untreated, cell lysates were prepared, and equal amounts of total protein were loaded and separated by SDS-PAGE. Src levels were determined by Western blot analysis. Values are presented as the mean ± SD (n = 4 experiments; *p < 0.05; t test). B, To determine the activity of c-Src, Oli-neu cells were treated with conditioned neuronal medium or were untreated, and the cell lysates were immunoprecipitated with anti-Src antibodies and subjected to kinase assays. Results are expressed as the mean ± SD of four experiments (*p < 0.05; t test). C, Primary oligodendrocytes (pOL) were cultured for 2–5 d, and levels of Src were determined at an early stage (cells with processes) and at a later stage of differentiation (cells with sheets) by quantifying immunofluorescence intensities. The specificity of the signal was demonstrated by preincubation with a blocking peptide (bp). Values are presented as the mean ± SE (n > 15 cells; **p < 0.01; t test). D, Oli-neu cells were cotransfected with plasmids encoding for PLP-myc and constitutively active c-Src-EGFP (Src ca). E, Primary oligodendrocytes were transfected to express constitutively active c-Src-EGFP (Src ca). Immunofluorescence analysis reveals colocalization of c-Src-EGFP with Lamp-1. Note also the perinuclear clustering of LE/Ls. F, Oli-neu cells were transfected with membrane-targeted GFP (ctrl) or the constitutively active c-Src (Src ca), treated with conditioned neuronal medium, labeled with LysoTracker red, and imaged by confocal microscopy. Relative motility of LE/Ls was determined as described in Figure 3 A. Values represent the mean ± SE (n > 20 cells; ***p < 0.001; t test). Scale bars, 10 μm.

Article Snippet: The following primary antibodies were used: hemagglutinin (HA) (16B12, mouse monoclonal; Covance, Berkley, CA), HA (rabbit polyclonal IgG; Abcam, Cambridge, UK), GFP (polyclonal rabbit; Abcam), GM130 (mouse monoclonal IgG1; BD Biosciences, Franklin Lakes, NJ), Lamp-1 (CD 107a, rat monoclonal; BD Biosciences), myc (rabbit polyclonal IgG; Upstate Biotechnology, Temecula, CA), myc (monoclonal IgG; Cell Signaling Technology, Denver, CO), O10 (monoclonal mouse IgM) ( Jung et al., 1996 ), RhoA (26C4, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA), and Src (36D10, rabbit monoclonal IgG; Cell Signaling Technology).

Techniques: Activity Assay, Incubation, SDS Page, Western Blot, Immunoprecipitation, Cell Culture, Immunofluorescence, Blocking Assay, Transfection, Labeling, Confocal Microscopy