Immunoprecipitation of GPCMV pentameric complex.

<p><b>(i)</b> Individual expression of pentameric complex components in epithelial cells transduced with recombinant adenovirus encoding either gHGFP, gLmCherry, GP129myc, GP131HA or GP133FLAG analyzed by western blot using respective primary antibodies: gH (anti-GFP); gL (anti-mCherry); GP129 (anti-myc); GP131 (anti-HA); GP133 (anti FLAG). Bands visualized with appropriate secondary antibody-HRP conjugate as described in materials and methods [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005755#ppat.1005755.ref036" target="_blank">36</a>]. Lys indicates defective adenovirus transduced lane and MI indicates mock cell lysate. <b>(ii)</b> Immunoprecipitation of the pentameric complex from epithelial cells transduced with all five recombinant defective adenoviruses encoding individual components (10 TDU/virus/cell). Samples were analyzed as total cell lysate as above or processed for immunoprecipitation assay. Immunoprecipitation was carried out with GFP trap as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005755#ppat.1005755.ref036" target="_blank">36</a>]. Individual proteins were detected as described for (i). Western blot lanes: 1–3 (anti-mCherry); 4–5 (anti-GFP); 6–8 (antiFLAG); 9–10 (anti-myc); 11–12 (anti-HA). Lanes 1 and 6 mock infected cell lysate. Lanes 2, 4, 7, 9 and 11 (total cell lysate). Lanes 3, 5, 8, 10 and 12 (immunoprecipitation). <b>(iii)</b> Control PC immunoprecipitation assay with GFP substituting for gHGFP. Epithelial cells transduced with defective recombinant adenoviruses encoding GFP, gLmCherry GP129myc, GP131HA and GP133FLAG followed by GFP immunoprecipitation and western blot for proteins as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005755#ppat.1005755.ref036" target="_blank">36</a>]. Western blot lanes: 1–4 (anti-GFP); 5–7 (anti-mCherry); 8–11 (anti-myc); 12–14 (anti-HA); 15–17 (anti-FLAG). Lanes 1 and 8 mock infected cell lysate. Lanes 2, 5, 9, 12 and 15 (total cell lysate). Lanes 3, 6, 10, 13 and 16 (flow through). Lanes 4, 7, 11, 14 and 17 (immunoprecipitation).