Fig. 3

Immunofluorescence and WB analysis of ST-IOWA cells transfected with PERV-C. A Immunofluorescence of SP-HA and RPep-HA transfected ST-IOWA cells using anti-HA-tag and anti-EnvC directed antibodies, 56 days p.t.. A1-4: ST-IOWA cells (ctr−), B1-4: PERV-C(5683) positive ST-IOWA cells (ctr+), C1-4: SP-HA and D1-4: RPep-HA transfected ST-IOWA cells. PERV-C Env proteins (red fluorescence) were detected for all cells except for the non-transfected control ST-IOWA. PERV-C Env-HA proteins were only observed for cells transfected with SP-HA or RPep-HA, as indicated by green fluorescence. B Detection of HA proteins by WB analysis in cell lysates of ST-IOWA cells transfected with SP-HA or RPep-HA harvested at day 56 p.t. (lane 1: ST-IOWA (ctr−), lane 2: SP-HA and lane 3: RPep-HA). Anti‐beta‐actin was used as loading control. PERV-C Env-HA precursor is expected at 72.1 kDa, its cleaved products SP-HA-SU at 52.3 kDa and TM-HA at 20.9 kDa. PERV-C Env-HA precursor proteins were detected for both, SP-HA and RPep-HA. SP-HA-SU shows only slight size variation compared to the precursor protein. For RPep-HA, both the precursor and the cleaved product were detected