Figure 5.

Recovery of COX-2 activity in immunoprecipitates. Cells were incubated for 6 h in the absence (control) or presence of 1 mM SIN-1 and/or 25 u ml⁻¹ IL-1α as indicated in the legend of Figure 1. (a) COX-2 activity. COX-2 protein was immunoprecipitated using an immunoaffinity column of monoclonal antibody selective for COX-2 and COX activity was assayed by incubating the immunoprecipitated proteins with 25 μM of arachidonic acid and haematin as described in the Methods section. After 15 min of incubation, the reaction was stopped and the PGE₂, one of the metabolites of PGH₂ was measured in the supernatant by EIA. Results are expressed as mean±s.e.mean of seven experiments. *P<0.05 with respect to IL-1α, unpaired t-test. (b) Detection of COX-2 protein after immunoprecipitation. The immunoprecipitates were recovered in Laemmli buffer and assayed for COX-2 expression by Western blot analysis. IgG corresponds to immunoglobulin recovered by immunoprecipitation.