Figure 2. Inhibition of clathrin-mediated endocytosis attenuates GMR signaling. (A) A schematic diagram for the GMR internalization assay. α rAb and β mAb stand for the rabbit and mouse antibodies recognizing GMRα and GMRβ, respectively; Anti-rAb and anti-mAb stand for Alexa flour^® 488- and Alexa flour® 555-conjugated secondary antibodies recognizing α rAb and β mAb, respectively. (B) HeLa cells transiently co-expressing GMRα (green) and GMRβ (red) were shifted to 37°C in medium containing or not containing GM-CSF (± GM) for 0–30 min and processed as outlined in (A), followed by image analysis using confocal microscope. (C) Liganded receptor endocytosis was allowed to proceed in HeLa cells transiently expressing GMRα and GMRβ in the presence of the indicated inhibitors. (D) Activation of JAK2, AKT, STAT5 and ERK in αβ-Ba/F3 cells pre-treated with MDC (300 μM) mβCD (5mM) or dynasore (80 μM) for 30 min prior to re-stimulation with hGM-CSF for the indicated time was analyzed by immunoblotting. Shown here is one representative result. (E) shows the average activation extent (phosphorylation) of each indicated protein (mean ± SEM) from three independent experiments. Only the p values of those treatment groups that are statistically significantly different from the control group at the same time point are as indicated (one way ANOVA, Tukey’s post test).