For visualization of GFP-tagged proteins transfected
Cyt387 were fixed on coverslips by incubation with 4% paraformaldehyde (PFA) for 10 min. Afterwards, DNA was counterstained with 4′-6-diamidino-2-phenylindole (DAPI, 300 nM) for 10 min at 37 °C. For immunofluorescence detection of HA/His-tagged c-Jun proteins cells were permeabilized using 0.2% Triton X-100/PBS. Cells were blocked for 1 h in 2% BSA/PBS and incubated for 1 h with the primary antibody (1:250 in 0.2%BSA/0.01%Tween/PBS). After 30 min incubation with the secondary antibody Alexa Fluor 546 (1:1000) (Invitrogen, Germany)
cells were mounted with Shandon Immu-Mount? (Thermo Scientific, Germany). Microscopic acquisition and analysis was performed using Volocity 5 from Perkin Elmer on an inverted epifluorescence microscope (Zeiss Axiovert S100, Germany) equipped with different objectives (HC PLAN APO 20×/0.7, PL FLUOTAR 40×/1.00, HCX PL APO 63×/1.40–0.6) and an X-Cite? 120 fluorescence illumination system (EXFO). By using Leica filter cube A, GFP and I3 the fluorophores DAPI, GFP and Alexa 546 were detected, respectively.
