Fig. 1. PMA/CaI-induced CD21 shedding in 293-CD21 cells. (A) 293-CD21cells were stained with the monoclonal anti-CD21 FITC-labeled antibody BL13. Expression and cell surface localization of the exogenous CD21 was verified by confocal microscopy. (B) Western blot analysis of 293-CD21 cells after 4 hours of stimulation with 10 µM PMA, 10 µM PMA + 1 µM CaI or the solvent control DMSO. 30 µg of protein lysate per lane were subjected to western blot analysis using the monoclonal anti-CD21 antibody BU32 (upper panel). The blot was stained with Ponceau Red to control the amount of loaded protein (lower panel). (C) Determination of sCD21 (measured as pg sCD21/ml/median fluorescence intensity of 293-CD21 cells) in cell culture supernatants from 293-CD21 cells by ELISA. Cell culture supernatants were collected after incubation of the cells for 4 hours with 10 µM PMA, 1 µM CaI, 10 µM PMA + 1 µM CaI or the solvent control DMSO. Data are mean of five independent experiments each measured in triplicate.