Figure 2. ACT-mediated endocytosis is dependent on Ca²⁺ influx, PKA activation, actin polymerization and raft-like microdomains integrity.

J774A.1 macrophages were pre-incubated for 30 minutes at 37°C with inhibitors of the toxin-induced calcium influx, La³⁺ (100 µM) and nifedipine (10 µM), with an inhibitor of PKA, KT5720 (10 µM), and with an inhibitor of actin polymerization, cytochalasin D (10 µM), before the addition of ACT (35 nM). Then, the surface staining of ACT, GM1 and the β2 integrin was measured using a flow cytometer (panels A and B). The effect of cholesterol depletion was assayed by pre-incubation of cells with methyl-β-cyclodextrin (10 mM) for 30 minutes, after which cells were washed and then incubated with ACT (35 nM) (panel C). Internalisation was measured by FACS as described in Materials and Methods. Data shown are the mean ± SEM of at least three independent experiments, with *p<0.05, **p<0.025 and ***p<0.001.