Figure 5
Expression and secretion of sGP2 from LASV GPC deletion variants. Cell extracts and supernatants from HEK-293T/17 transfected with LASV GPC deletion variants were analyzed for the expression and secretion of GP1 and GP2 proteins. Ten micrograms of total protein from transfected cells were resolved on SDS-PAGE gels, blotted onto nitrocellulose membranes and probed with FLAG M2 mAb (A). Twenty μL of supernatant from the corresponding samples were similarly resolved, blotted, and probed with FLAG M2 mAb (B) or anti-LASV GP1 mAbs (C). (Lane 1) control plasmid pcDNA3.1(+):intA, (lane 2) GPCΔTMΔIC, (lane 3) GPCΔTMΔIC-FLAG, (lane 4) GPCΔTM-FLAG, (lane 5) GPCΔ59–249 ΔTMΔIC, (lane 6) GPCΔ59–249ΔTMΔIC-FLAG, (lane 7) GPCΔ59–249ΔTM-FLAG, (lane 8) GPC-FLAG, (lane 9) sGP1-FLAG. SeeBlue® Plus2 pre-stained molecular weight markers (M), with sizes (kDa) are shown to the right of the panel. Designations for the constructs that generated the intracellular expression pattern in lanes 6A and 7A are displayed to the left of panel A. Similarly, designations that generated the secreted expression pattern in lanes 3B and 4B are displayed to the left of panel B. Positions of monomer (m), dimer (d), and trimer (t) forms of the GP1 protein are indicated by arrows. Similarly, the position of monomeric (m) and dimerized (d) forms of sGP1 (right), and polyprotein (poly) species consisting of sGP1+sGP2 (left) in panel B are indicated by arrows.