Overexpression of GPAT4 in Cos-7 cells increases NEM-sensitive GPAT activity. Cos-7 cells were transfected with pcDNA3.1 vector. Agpat2, Gpat1, or Gpat4. A: Immunoblot of Cos-7 cell total membrane fractions against the FLAG epitope (two independent transfections). B: GPAT specific activity (SA.) in Cos-7 total membrane fraction (vector, n = 13; GPAT4, n = 12; GPAT1, n = 4; AGPAT2, n = 2). * P < 0.0001 compared with vector; # P < 0.0001 compared with GPAT4; § P < 0.0001 compared with GPAT1. C: AGPAT specific activity in Cos-7 cell total membrane fraction (n = 3). * P < 0.01 compared with vector. D: Radiolabeled glycerolipid products from GPAT assays of Cos-7 cell total membranes were separated by TLC and quantified as described in Methods (average of two independent assays with three separate samples per assay). DAG, diacylglycerol; LPA, lysophosphatidic acid; PA, phosphatidic acid. * P < 0.05 compared with vector. E: Total GPAT activity in Cos-7 total membrane preparations with different acyl-CoA substrates (60 μM) (average of two independent experiments). Data are shown as means ± SEM.