Figure 2

Stabilization of the co-expressed F and G proteins by in situ protein cross-linking. (A) Cells co-transfected with pCAGGS/F-cmyc and pCAGGS/G-FLAG, surface-biotinylated and lysates immunoprecipitated (IP) with anti-cmyc and or anti-FLAG probed with streptavidin-HRP. (B) LLMCK2 cells were either mock-infected or infected with HMPV. At 7 days post-infection the cells were surface biotinylated and lysates were IP with anti-G, or anti-F. The various G and F protein species are indicated. (C and D) Cells were co-transfected with pCAGGS/F-cmyc and pCAGGS/G-FLAG, IP with anti-cmyc or anti-FLAG and immunoblotted (IB) with (C) anti-cmyc or (D) anti-FLAG. (E-H) Cells were co-transfected with pCAGGS/F-cmyc and pCAGGS/G-FLAG and treated with increasing concentrations of DSP (0-1 mM). Detergent extracts were IP with anti-cmyc and IB with either (E) anti-FLAG or (G) anti-cmyc, or IP with anti-FLAG and IB with (F) anti-cmyc or (H) anti-FLAG. (I and J) pCAGGS/F-cmyc and pCAGGS/G-FLAG co-transfected cells were treated with 0.1 mM DSP and a detergent extract prepared. This was clarified and loaded onto a 5-30% sucrose gradient. The gradient was fractionated and each fraction (I) IP with anti-cmyc and IB with anti-FLAG, or (J) IP with anti-FLAG and IB with anti-cmyc. The various monomeric and oligomeric F and G protein species are indicated. High molecular mass F proteins are highlighted with “*”.