Measurement of PLA activities in Flp-INTRex293 cell culture. (A) PLA2 activity. Isogenic Flp-INTRex293 cells that express a single copy of GPR34 or its mutants, together with the parental cell line, were incubated with the substrate and liposome mix, and monitored for PLA2 enzymatic activity by using the EnzChek Phospholipase A2 Assay Kit. (B) Combined PLA1 and PLA2 activities. Isogenic Flp-INTRex293 cells that express a single copy of GPR34 or its mutants, together with the parental cell line, were incubated with 16:0-18:1 PS, and their culture supernatants were monitored for free fatty acid release at the indicated times by measuring their conversion to coenzyme A derivatives. Details are provided in the Materials and methods section for these assays. For each assay, the data (mean ± standard deviation) presented are from 3 independent experiments. Statistical differences among the various cell lines were analyzed by using a linear regression model, with significant differences indicated. (C) Confirmation of PLA1 in culture supernatant of Flp-INTRex293 cells by western blot analysis. Control: the parental Flp-In T-REx-293 cell line. Wild-type (WT) or various mutations indicated: the derived stable expression cell line with single copy of WT or mutant GPR34.