Fig. 4.

Inhibition of the E1 ubiquitin-activating enzyme blocks G-CSFR internalization. G-CSFR surface expression was quantified from multiple confocal images using LSM5 software to measure the Cy5 fluorescence intensity over the outer 0.35 mM of individual ts20 cells transfected with the WT or Δ716 G-CSFR. The cells were stimulated with G-CSF (100 ng/mL) for 1h at 4°C, then incubated at 30°C or shifted to 42°C to inactivate the E1 enzyme. Cells were fixed and sequentially incubated with biotin-conjugated mouse anti-human G-CSFR antibody (CD114, BD PharMingen) and Streptavidin-Cy5 (Caltag) to detect the G-CSFR. (A) Yellow arrow (left panel) depicts the region used to measure fluorescence intensity from a representative image of a cell expressing the Δ716 G-CSFR incubated for 30 min at 30°C. Histogram (right panel) shows the fluorescence intensity of Cy5 (red) and DAPI (blue) staining in the region corresponding to the yellow arrow. (B) Bar graphs show the mean Cy5 fluorescence intensity corresponding to G-CSFR surface expression on cells stimulated with G-CSF and incubated for the indicated times at either 30°C or 42°C.