The majority of GP5–HA molecules in transfected cells is cleaved at site 2.

<p>MARC-145 cells were transfected with variants of GP5–HA or with empty plasmid (Ø), subsequently lysed and assessed by SDS-PAGE and Western blot (anti-HA tag) before (A) and after (B) PNGase F digestion to remove glycans. wt: GP5–HA wildtype; uncl.: Signal peptide (SP) cleavage completely blocked by mutation A26F, A29Y, A31F; cl.1: SP cleavage possible at site 1 (A26|V27) only (mutation A29S, A31Y); cl.2: SP cleavage at site 2 (A31|S32) only (mutation A26F, A29S). Black arrow: GP5–HA with cleaved SP, carrying three glycans, and/or GP5–HA comprising SP and two glycans; black arrow with plus sign: GP5–HA, SP cleaved, four glycans; white arrow: GP5–HA unprocessed/deglycosylated and containing the SP; grey and black arrowhead: GP5–HA with SP cleavage at site 1 or 2, respectively. (C), limited PNGase F digestion of GP5–HA uncl., performed and labeled as in Fig. 4. The protein carries two glycans.