ARHGAP25 regulates phagocytosis of ARHGAP25-silenced cells. (A) Expression of ARHGAP25 in PLB-985 cell line during the 7-day differentiation to neutrophils. (B) Western blot signal is significantly reduced in ARHGAP25-silenced PLB cells compared with control shRNA treated and nontreated cells. (C) Western blot analysis of ARHGAP25-silencing by siRNA in human macrophages. In Western blot experiments, staining for β-actin was used as loading control. (D) Yeast cells were labeled with Cell Tracker Green and were opsonized with pooled human serum (empty columns) or heat-inactivated human serum (hatched columns) for 1 hour. After 10 minutes' co-incubation, the number of PLB cells that had engulfed one or more yeast particles was counted by flow cytometer and normalized to 100 cells. Mean + SEM of 7 separate experiments is shown (*P < .05 vs control shRNA treatment). (E) Pooled serum opsonized yeast uptake of ARHGAP25-silenced human macrophages. The number of macrophages phagocytosed one or more yeast particles was calculated by flow cytometer and normalized to control siRNA treated cells. Mean + SEM of 3 separate experiments is shown. (F-G) F-actin level in ARHGAP25-silenced PLB cells, compared with control shRNA-treated cells. F-actin was stained with Alexa-488–labeled Phalloidin, F-actin level was measured with flow cytometer. Panel F shows a representative result of the experiment. In panel G mean + SEM of 16 (control shRNA-treated) and 13 (ARHGAP25-silenced) separate experiments are shown.