Fig. 5. Suppression of M-CSF signaling by HA. (A) BMMs were serum-starved, pretreated with HA (1 µg/ml) for 30 minutes and stimulated with M-CSF (300 ng/ml). Cell lysates were immunoblotted with phosphorylation-specific antibodies to detect the activation of ERK (a), JNK (b), and p38 (c). The same membranes were stripped and reprobed to detect total levels of each MAPK. The relative levels of phosphorylated forms were determined by densitometry. (B) BMMs were serum-starved and pretreated with HA (1 µg/ml) for 30 minutes. The cells were loaded with DCF-DA and stimulated with M-CSF (300 ng/ml) for 5 minutes. The DCF fluorescence was detected by confocal microscopy. The inbox in the upper left panel shows single-cell fluorescence. The average of the mean fluorescence intensity of several fields is presented as a histogram. ^*P<0.05 compared with HA-untreated group. (C) BMMs were serum-starved, pretreated with HA (1 µg/ml) for 30 minutes and stimulated with M-CSF (300 ng/ml) for 5 minutes. Total cell lysates were prepared and subjected to the Rac activity assay (top). In the bottom panel, stimulated cells were lysed and cytosolic and membrane fractions were immunoblotted with anti-Rac antibody. The same membranes were reprobed with anti-ß-actin and anti-flotillin antibodies to ensure comparable amounts of loading.