Erk1/2 phosphorylation analysis

Fresh isolated neutrophils (1 × 10⁶ cells/ml) were resuspended in Ca²⁺-containing HBSS and stimulated with drugs as indicated in the text for 30 min at room temperature. Then, the cells were activated with 100 nM fMLP for 1 min after pre-warming for 5 min at 37 °C, followed by the addition of a 5-fold volume of ice-cold HBSS and storage on ice to stop the reaction. The cells in Ca²⁺-free HBSS were used as negative controls for Ca²⁺ influx. For Erk1/2 phosphorylation analysis, neutrophils were lysed in RIPA with phosphatase inhibitor cocktail 3 and PMSF. Equal amounts of proteins from lysed cells or were quantified and then subjected to SDS-PAGE (4.8% stacking gel and 10% resolving gel). The next western blot operation is similar to the one mentioned above except rabbit monoclonal antibodies against phospho-Erk1/2 (1:1000, 4370S, CST, Danvers, MA, USA) and Erk1/2 (1:1000, 4695S, CST, Danvers, MA, USA) were used as the primary antibodies.