Fig. 5. Interaction between the structural Gag and the cytoplasmic light chain of dynein LC8. (A) Cos6 cells were co-transfected with pSG-Gag and pGFP-LC8, and analyzed 20 hours later. As shown in this confocal section, 97% of Gag co-localizes with GFP-LC8 at the MTOC [measured using Lasersharp software (BioRad)], whereas GFP-LC8 is evenly distributed in the cytoplasm when expressed alone. Nuclei are stained with DAPI. (B) A direct interaction between LC8 and Gag was observed by immunoprecipitation experiments followed by western blotting when 293T cells were co-transfected with the expression vectors, whereas the L171G mutation abolishes this interaction. Anti-Gag and anti-Flag antibodies do not precipitate LC8 or HFV Gag, respectively, when these proteins are expressed alone. A similar interaction was observed with the M2 mutant (GFP-Gag150-250) but not with the M9 mutant, which lacks the domain involved in MTOC targeting.