Fig. 4.

Sensitive detection and quantification of substrate-specific subcellular phosphatase activities. A–F, SE phosphatase assays were performed with 2-fold serially diluted AC16-CAR extracts starting at 25,000 (A–C), 50,000 (D–E), or 100,000 (F) cell equivalents. G–L, NE phosphatase assays were performed with 2-fold serially diluted extracts starting at 17,000 (G–I, K) or 51,000 (J, L) cell equivalents. NE fractions for phospho-MK2 (J) and phospho-CREB (K) were collected from cells stimulated with TNF for 30 min. Black arrows denote the optimized extract concentration for each assay. Phosphatase activity data were regressed against cell input with a four-parameter logistic regression curve. Data are shown as the means ± standard error of n = 4 assay replicates.