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. 2015 Aug 12;10(8):e0135567. doi: 10.1371/journal.pone.0135567

Fig 4. GPCMV gM /gN complex formation and immunoprecipitation (IP) assays.

Fig 4

All IPs were performed with GFP-Trap (ChromoTek) as described in materials and methods to immunopreciptiate proteins that interacted with gMGFP or GFP control. (i) gMGFP and gNmCherry co-expression and IP. Lanes 1 and 4 are total cell lysate of gMGFP and gNmCherry, respectively. Lanes 3 and 6, IP reactions. Lanes 2 and 5, control mock (MI) cell lysate. For gM detection, anti-GFP antibody (lanes 1–3). For gN detection, anti-mCherry antibody (lanes 4–6). (ii) Control GFP IP. GP84 protein tagged with GFP [51] was co-expressed with gN(f)FLAG. Lanes 1 and 4 total cell lysate for GP84GFP and gN(f)FLAG respectively. Lanes 3 and 6, IP reactions for GP84GFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock control (MI). (iii) gMGFP and gN(f)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysates of gMGFP and gN(f)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(f)FLAG transfected cells. Lanes 2 and 5 mock (MI) control cell lysate. 6. Detection for gN(f)FLAG by anti-FLAG antibody. (iv). gMGFP and gN(s)FLAG co-expression and IP. Lanes, 1 and 4 are total cell lysate of gMGFP and gN(s)FLAG transfected cells respectively. Lanes 3 and 6, IP reactions for gMGFP and gN(s)FLAG transfected cells. Lanes 2 and 5, mock control (MI). Detection for gN(s)FLAG by anti-FLAG antibody. Specific protein bands are indicated by an arrow. In gMGFP expressing cells a second higher MW protein (100 kDa) was detected and labelled x. All gels (4–20%) SDS-PAGE included a lane for a kDa ladder (MagicMark Protein Standard, Life Technologies). Ladder lanes not shown.