Figure 4. Demonstration of N-and O-glycosylation.

A. Demonstration of N-and O-glycosylation of spectrin by enzyme deglycosylation. Equal amount (5 µg) of purified spectrin_VL and spectrin_N was treated with neuraminidase from Arthrobacter ureafaciens to remove the terminal sialic acids and subsequently desialylated spectrin_VL and spectrin_N was incubated separately with N-glycosidase F, O-glycosidase or a combination of N- and O-glycosidase as indicated. Spectrin_VL/N before and after the respective enzyme treatments were analyzed by SDS-PAGE as described in Materials and Methods. B. Demonstration of sialylation, N- and O-glycosylation in 60 kDa fragment. Gel-eluted purified 60 kDa fragment (1.0 µg) was initially desialylated with Arthrobacter ureafaciens neuraminidase overnight at 37°C. Subsequently the desialylated 60 kDa fragment was treated separately with N-glycosidase F, O-glycosidase F or a combination of both and analyzed by SDS-PAGE (7.5%) along with the untreated protein as described in Materials and Methods. Gel was stained with silver staining method. Lane M shows molecular weight standards. C–D. Demonstration of N- and O-glycosylation by lectin binding with ¹²⁵I-spectrin_VL/N. Fixed concentrations of ¹²⁵I-spectrin_VL/N were processed separately to demonstrate their binding with several Sepharose/agarose bound ConA, RCA, HPA, UEA, DBA and Jacalin lectins (25 µl bead volume) having different sugar-linkage specificity as described in Materials and Methods. E–F. Demonstration of N- and O-glycosylation by lectin binding with DIG-glycan. E. Equal amount (2.0 µg) of spectrin_VL and spectrin_N was dot blotted on NC-paper and analyzed by DIG-glycan and differentiation kit using several lectins (GNA, PNA, DSA) following manufacturer's protocol. F. Representative bar graph of densitometric scores of corresponding spots.