Figure 2

Analysis of MNF partners by co-immunoprecipitation. Ten mm plates of BGMK cells (Baby green monkey kidney) were infected with MYXV-GFPMNF (1), wild-type MYXV (2) or MYXV-GFP (3), at m.o.i 3. The latter has been obtained by insertion of GFP gene, under control of the P7.5 poxviral promoter, in the thymidine kinase locus. 24 hours post-infection, cells were washed in PBS and lysed with hypotonic buffer (10 mM HEPES pH7.9, 150 mM NaCl, 600 mM KCl, 0.5% NP40 and proteases inhibitors). Lysates were cleared by centrifugation and the supernatants incubated with μMACS anti-GFP MicroBeads. Washes and elution were performed according to manufacturer's instructions. Eluates were loaded on SDS/PAGE gels and analyzed by Simply Blue SafeStain (A) or by western blot (B). A. The 3 bands specific of MYXV-GFPMNF precipitation (a b and c) were analyzed by mass spectrometry. B. Western blot analysis of lysates with rabbit anti-GFP, rabbit anti-Cullin-1, rabbit anti-Skp1 or mouse anti-ubiquitin antibodies. Secondary antibodies were from anti-rabbit or anti-mouse WesternBreeze Chemiluminescent Kit, respectively. c and →: GFPMNF, *: GFP, a and b: Cullin-1 bands, and ⇒: Skp 1 band.