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Fig. 6. SCR 16 is required for CD21 shedding. (A) Schematic presentation of CD21 constructs. Several CD21 cDNAs containing exon 16 (B-E) and one without (A) were cloned into the eucaryotic expression plasmid pEGFP-C1 as described in Materials and Methods. The resulting CD21 proteins are schematically shown on the right. (B) Verification of the expression and cell surface localization of the different CD21 mutant proteins expressed by 293 cells, stable transfected with the constructs shown in Fig. 6A. Cells were stained with anti-CD21-RPE and subjected to flow cytometry analysis. One clone of each cell line is shown (293-CD21-A, violet; -B, orange; -C, magenta; -D, light blue; -E, green). Untransfected 293 cells (blue) were stained as a control. (C) Determination of the amount of sCD21 in cell culture supernatants after stimulation of the cells for 4 hours at 37°C with 10 µM PMA + 1 µM CaI, 200 µM pervanadate, 5 mM NAC or 5 mM GSH. Cell culture supernatants were collected and sCD21 was measured by ELISA (values expressed as pg sCD21/ml/median fluorescence intensity of respective clones). Three clones of each construct were tested and gave the same results. Data are means of two independent experiments of a single clone of each cell line.
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