|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 2. Targeting of P2X4 receptors to lysosomes mediated by N-terminal dileucine and C-terminal tyrosine-based motifs. (A) Confocal images of wild-type and mutant P2X4 receptors containing an extracellular AU5 epitope expressed in NRK cells. Surface proteins were labelled by incubating live cells for 30 minutes with extracellular antibody against AU5 at 37°C. Cells were then fixed, permeabilized and total receptors labelled with antibody against P2X4. Bars, 10 µm. (B) Histogram showing the ratio of labelled to total fluorescence normalized to the P2X4 (AU5) receptor. n=22–39 per construct. For P2X4 (AU5), only cells showing some detectable surface labelling were analyzed and so the ratio is likely to be an overestimate relative to the mutant receptors. (C) Graph showing the effects of mutating dileucine and tyrosine motifs on the internalization of P2X4 receptors. NRK cells expressing vectors encoding P2X4 dileucine and tyrosine mutant proteins were incubated with extracellular antibody against AU5 at 37°C for 30 minutes. Cells were fixed, surface receptors detected by secondary antibody, permeabilized and internalized receptors detected with a different secondary antibody. n=21–43 cells per construct. (D) Surface expression of P2X4 receptors with mutations in dileucine and tyrosine motifs was measured by biotinylating surface proteins, purifying with streptavidin beads and probing with antibody against P2X4 by western blot analysis. The `Total' gel shows dilutions of total cell lysate for each construct. (E) Effect of dileucine and tyrosine mutations on the lysosomal distribution of P2X4 receptors. GFP-tagged constructs (green) were expressed in NRK cells and were stained with antibody against LAMP-1 (red). The histogram shows the values of Pearson's correlation coefficient calculated using Zeiss LSM software. n=16–28 per construct. Experiments were repeated three times and the data shown are from one representative experiment; error bars indicate means+s.e.m. (***=P<0.001). Bars, 10 µm.
|
