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. 2013 Jun 6;8(6):e65548. doi: 10.1371/journal.pone.0065548

Figure 4. Limited PNGase F digestion of GP5–HA to show modification with three glycans.

Figure 4

MARC-145 cells were transfected with the GP5–HA wt construct. Aliquots of the cell lysates were treated with decreasing concentrations of PNGase F as indicated or left undigested (Ø), then analyzed by SDS-PAGE and Western blot (anti-HA tag). The number of carbohydrates cleaved by PNGase F decreases with decreasing concentration. This causes a ladder-like appearance of bands that allows counting of the total number of carbohydrates linked to GP5–HA (arrowheads). The band denoted with the asterisk must not be counted since it is also present in untreated GP5–HA and probably represents unprocessed/non-translocated protein (see also Fig. 3C, white arrow).