Figure 2.

β2 undergoes complex N-glycosylation and is sialylated at Asn-42. MDCK cells were transiently transfected with the SCN2B-yfp vector to express WT or partially or fully unglycosylated β2 and grown for 1 day in wells. Representative Western blots are shown with the same amount of protein lysate loaded into each lane. Note that immature (unprocessed) β2 is clearly discernible from the slowly migrating mature form (compare with Fig. 1). A, denatured protein from cell lysates was treated overnight at 37 °C with Endo H to cleave off immature N-glycans (faster-migrating band) in β2. B, lysates were treated overnight at 37 °C with NA to cleave off all terminal sialic acids. The upper band displays a slight increase in mobility in WT and single and double mutants not including the N42Q mutation (red type). Blots for actin are included as loading controls. Molecular mass markers are in kDa. The dividing line in B separates different blots (taken from the same exposure) conveniently put together for clear display.