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Results: 5

1.
Figure 4

Figure 4. From: Neuronal polarity: Essential role of protein-lipid complexes in axonal sorting.

Changes in Thy-1 insolubility in stage 5 neurons after inhibition of sphingolipid synthesis with fumonisin B1. Neurons were maintained in culture for 8 days in the absence (A and B) or presence (C and D) of 25 μM FB1. Then the cells were extracted in 1% Triton X-100, washed, fixed, and processed for immunofluorescence by using an antibody against Thy-1. (A and B) Phase contrast and Thy-1 staining, respectively, of control detergent-extracted neurons. The detergent is not able to solubilize the protein from the axon of the cells. (C and D) Phase contrast and Thy-1 staining, respectively, of FB1-treated, detergent-extracted neurons. In this case the detergent is able to remove the protein.

Maria Dolores Ledesma, et al. Proc Natl Acad Sci U S A. 1998 March 31;95(7):3966-3971.
2.
Figure 1

Figure 1. From: Neuronal polarity: Essential role of protein-lipid complexes in axonal sorting.

Axonal distribution of HA in stage 5 neurons. (A) FPV-infected stage 5 neuron stained with an antibody against the dendritic marker MAP2; the axon (ax) appears devoid of labeling. (B) The same infected neuron was coimmunostained with an antibody against HA. The labeling is mainly present in the axon (ax), leaving the dendrites (de) almost unstained. This was quantified by binarizing the image in B and choosing at random similar areas in dendrites and axons as is shown in C. The number of pixels was analyzed and normalized to the same area size in each process. (D) Percentage of HA present in dendrites versus axons in 12 different neurons. The mean value for the distribution of HA is 78% in the axons and 22% in the dendrites.

Maria Dolores Ledesma, et al. Proc Natl Acad Sci U S A. 1998 March 31;95(7):3966-3971.
3.
Figure 3

Figure 3. From: Neuronal polarity: Essential role of protein-lipid complexes in axonal sorting.

Thy-1 distribution and insolubility in stage 5 neurons. (A) Double immunofluorescence of a stage 5 neuron using an antibody against the dendritic marker MAP2 (in red) and against Thy-1 (in green). The axonal distribution of surface Thy-1 is evident. (B) Quantitative analysis of Thy-1 distribution from three different experiments (1, 2, and 3). In each experiment an average number of eight dendrites (MAP2 positive) and eight axons were examined. The mean values of the Thy-1 labeling in axons versus dendrites are shown by histograms as a percentage of the total Thy-1 labeling observed in each experiment. Eighty to 95% of Thy-1 is axonal. (C) The soluble (S) and insoluble (P) fractions after extraction of cell lysates with 1% Triton X-114 at 4°C were analyzed by Western blot by using an antibody against Thy-1. A significant amount of the protein (33%) remains insoluble.

Maria Dolores Ledesma, et al. Proc Natl Acad Sci U S A. 1998 March 31;95(7):3966-3971.
4.
Figure 5

Figure 5. From: Neuronal polarity: Essential role of protein-lipid complexes in axonal sorting.

Missorting of the axonal Thy-1 but not the dendritic GluR1 occurs in stage 5 neurons after incubation with fumonisin B1. Neurons were maintained in culture for 8 days in the absence (A) or presence (BD) of 25 μM FB1. Then the cells were processed for double-immunofluorescence microscopy by using antibodies against MAP2 (AC) and Thy-1 (A and B) or GluR1 (D). MAP2 is restricted to the dendrites and serves as a marker for these processes in control and treated cells. (A and B) Overlay of MAP2 (in red) and Thy-1 (in green) staining in control (A) and FB1-treated (B) neurons. In control cells Thy-1 is present specifically in the axons (indicated by arrowheads) and does not colocalize with the dendritic marker. In FB1-treated neurons the axonal-exclusive Thy-1 labeling (in green; axons are indicated by arrowheads) is lost and dendrites appear yellow because of the colocalization of MAP2 and Thy-1. The dendritic distribution of the glutamate receptor 1 in control cells (not shown) does not change after FB1 treatment, and the protein (D) colocalizes with the dendritic marker MAP2 (C) whereas the axon of the cell (arrowheads) remains unstained.

Maria Dolores Ledesma, et al. Proc Natl Acad Sci U S A. 1998 March 31;95(7):3966-3971.
5.
Figure 2

Figure 2. From: Neuronal polarity: Essential role of protein-lipid complexes in axonal sorting.

HA insolubility in stage 5 neurons. (A) HA insolubility during the biosynthetic pathway. FPV-infected stage 5 neurons were metabolically labeled. After chases of 0, 90, or 180 min the cells were extracted with 20 mM CHAPS at 4°C and centrifuged. The amount of HA present in supernatants (S) and pellets (P) was determined by 10–20% SDS/PAGE and autoradiography. The mature form of HA becomes more and more insoluble with time. The lower-molecular-weight band present in the supernatants corresponds to the immature form of HA that remains in the ER. (B) HA insolubility on the surface. FPV-infected stage 5 neurons were biotinylated, and were CHAPS extracted. The resultant supernatants (S) and pellets (P) were analyzed by SDS/PAGE and autoradiography. Almost all surface HA is detergent-insoluble (87%). The graphic shows the data expressed as percent of the total surface HA and mean values ±SD from three different experiments. (C) Isolation of detergent-insoluble complexes. TX-100 extracts of stage 5 neurons infected with FPV (HA), noninfected (Glu Rc), or infected with SFV (E2) were prepared at 4°C and centrifuged to equilibrium in a sucrose gradient. Fractions of equal volume were collected and the amount of sucrose was determined with a refractometer (numbers at the bottom indicate percentage of sucrose). The fractions were analyzed by Western blot by using the following antibodies: anti-HA (HA), anti-AMPA glutamate receptor subunit 1 (Glu Rc), and anti-E2 (E2). HA from stage 5 cells is found in the lighter fractions (24–9% sucrose), revealing its interaction with lipids, whereas the dendritic proteins glutamate receptor and the viral E2 are found in the high density fractions (37 and 39% sucrose).

Maria Dolores Ledesma, et al. Proc Natl Acad Sci U S A. 1998 March 31;95(7):3966-3971.

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