Lectin blot analysis of glycoconjugates isolated from human CLA⁺ T cells treated with glycosylation inhibitors.

Cell lysates were prepared from human CLA⁺ T-cell cultures treated with diluent control (PBS), tunicamycin (0.02 mM), swainsonine (0.23 mM), 4-F-GlcNAc (0.05 mM), BAG (2.0 mM), or GlcNAc (5.0 mM, negative control) for 30 hours. Lysates (25 μg per spot) were spotted onto permeabilized PVDF membrane, blocked in FBS for 1 hour at RT, and blotted with AP-conjugated ConA (2.0 μg/mL PBS), WGA (0.5 μg/mL PBS), or LEA (1.0 μg/mL PBS). Blots were then washed 3 × with PBS/0.1% Tween-20 and developed with Western Blue stain. Please note the reduction in ConA, WGA, and LEA staining intensity of lysates from tunicamycin-treated cells; LEA staining was also diminished, yet WGA staining was increased in lysates from 4-F-GlcNAc–treated cells. ConA staining of lysates from swainsonine-treated cells was slightly increased.