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. 1999 Apr;10(4):1163–1178. doi: 10.1091/mbc.10.4.1163

Figure 3.

Figure 3

Characterization of the C5aR-GFP chimera. (A) C5aR–GFP can mediate chemotaxis. HEK293 cells stably expressing either the C5aR or the C5aR–GFP fusion protein were subjected to a migration assay in a 48-well Boyden chamber, as described previously (Neptune and Bourne, 1997). At the end of the assay, cells adhering to the lower side of the porous filter were fixed, stained, and counted using a microscope. Results are expressed as the number of cells counted in a high-power field (HPF) (200×) and correspond to the mean ± SD of four determinations. Similar results were obtained in four other experiments. Absolute numbers of migrated cells in the experiment shown do not reflect different abilities of the two cell types to migrate toward C5a; instead, the absolute numbers reflect the numbers of each cell type used in each well (22,500 and 37,500 cells expressing the C5aR–GFP or the C5aR, respectively). (B) Integrity of the C5aR–GFP fusion protein in differentiated PLB-985 cells. PLB-985 cells were transduced with a retrovirus carrying C5aR–GFP as described in MATERIALS AND METHODS. Transduced cells were selected and GFP-positive cells were sorted by FACS. Cell extracts were prepared from 5 × 106 differentiated cells as described in MATERIALS AND METHODS, resolved on 8% polyacrylamide, and transferred onto a polyvinylidene difluoride membrane. GFP and the C5aR–GFP were then revealed with a monoclonal GFP antibody. Lane 1, extract from 105 GFP-expressing cells; lanes 2 and 3, extracts from two different aliquots, each representing 3 × 105 C5aR-GFP–expressing cells. (C) Binding of [125I]-C5a to PLB-985 cells. Differentiated GFP-expressing cells (squares) and C5aR-GFP–expressing cells (circles) were incubated at 4°C for 5 h with the indicated concentrations of [125I]-C5a (0.06–3.6 nM). Nonspecific binding was evaluated in the presence of 100 nM nonradioactive C5a. Incubations were terminated by rapid filtration though GF/C filters. In the experiment shown, nonlinear regression analysis of the binding data yielded Bmax values of 68,400 sites/cell and 47,600 sites/cell for C5aR–GFP and GFP-expressing PLB-985 cells, respectively. Similar results were obtained in two additional experiments.