Fig. 4.

The effect of nedocromil and dexamethasone on the disposition of Anx-A1 in U937 cells transfected with an Anx-A1–GFP construct. (Panel A, upper figure) The Western blot, probed with an anti-GFP antibody, shows that only GFP protein (27 kDa) was seen in U937 cells transfected with the mock plasmids E-1, E-2 and E-3 whereas in U937 cells transfected with Anx-A1–GFP constructs G-1 and G-2 a 64 kDa band corresponding to the GFP–Anx-A1 conjugate was observed. Clones G-1, G-2, E-1 and E-3 were used for this study. (Lower figure) This blot shows the complementary experiment to that in panel A, where the samples were probed with the anti-Anx-A1 antibody. (Panel B) In these experiments, the plasma membrane of U937 cells transfected with the Anx-A1–GFP construct, was stained red with Alexa Fluor^© 594 WGA dye and the cells subjected to various drug treatments protocols. The four columns represent the information from the two colour channels, the merged channel and a magnified (90×) section of the membrane. (Row a) The situation when the transfected U937 cells are treated with vehicle alone. Anx-A1 is mainly contained within the cytoplasmic compartment and the plasma membrane stains red. (Row b) There appears to be little evidence of co-localisation after treatment with 5 nM nedocromil alone. (Row c) Treatment with dexamethasone alone 2 nM clearly promotes co-localisation of Anx-A1 and membrane phospholipids. (Row d) Treatment with dexamethasone and nedocromil in combination leads to a striking co-localisation of Anx-A1 with the membrane and evidence of release of GFP-tagged protein into the external medium. Magnification 90×. This figure is representative of three independent experiments.