Pro q emerald 300 glycoprotein gel stain kit

Experiments were carried out as previously described.^{36 (link)} Briefly, HEK293 cells were treated with 1 or 5 mM glucosamine (GlcN; G4875; Sigma) for 48 h and harvested as described above. Equal amounts of cell lysates were separated by SDS–PAGE. Gels were stained for general glycosylation of proteins using the Pro-Q Emerald 300 Glycoprotein gel stain kit (P21857; Thermo Scientific). To specifically stain for N-glycosylated proteins, proteins were transferred to nitrocellulose membranes, blocked with 5% nonfat milk for 1 h, and probed overnight with the concanavalin A–biotin conjugate (30 μg/mL) (C2272; Sigma). Immunoblots were washed three times (10 min each) in PBS containing 0.1% Tween 20 and incubated with horseradish perioxidase-conjugated avidin (1:20000) (1706528; Bio-Rad). Blots were washed three times (10 min each) in PBS containing 0.1% Tween 20, developed in ECL reagent (Millipore) for 2–5 min, and visualized with MultiImage III FluorChem Q (Alpha Innotech). Subsequently, gels and blots were stained for total protein using EZBlue Gel Staining Reagent (G1041; Sigma). Quantifications of signal intensity were performed using ImageJ software.