Fig. 2. Subcellular fractionation on continuous sucrose gradients revealed the accumulation of Golgi-glycosylated TCRα in post-ER fractions. HeLa cells were transiently transfected with TCRα, and collected without treatment (A) or after incubation in the presence of bafilomycin A1 (10^–6 M) at 37°C for 6 h (B). PNSs from the transfected cells were separated on continuous sucrose gradient (20–50%). Twelve fractions were obtained from each sample (fraction 1, top; fraction 12, bottom). An aliquot of each fraction was analyzed by SDS–PAGE. The distribution of TCRα, Golgi p58 and calnexin was determined by direct western blotting. While a 38 kDa core-glycosylated TCRα was localized in the ER fractions, a 43 kDa Golgi-glycosylated TCRα was found in the post-ER fractions. Aliquots of the ER [fraction 9 in (A)] and the post-ER [fraction 2 in (B)] fractions were treated with endo H and analyzed by SDS–PAGE. The membrane was immunoblotted using a rabbit anti-murine TCRα antiserum (C). A 43 kDa TCRα (p43) in the post-ER fraction acquired resistance to endo H digestion, while a 38 kDa TCRα (p38) in the ER fraction remained sensitive.