Figure 4

Intracellular localization of Rex protein isoforms. a IF/LSM of Rex isoforms in HLtat cells transfected with pMH-Rex, pMH-Rex_a, pMH-Rex_b and pMH-Rex_c plasmids. The Rex signal is visualized in green, and propidium iodide (PI), used as a nuclear marker, is visualized in red. b Quantitative analysis of the Rex (X axis) and PI (Y axis) fluorescence per pixel (represented by dots in the scatter plot). c The ‘‘Histogram’’ software tool was used to measure the Colocalization Index (CI), indicating the fraction of Rex-positive pixels that were also PI-positive; bars represent mean CI values and standard error bars for at least 50 cells. Plasmids encoding Rex, Rex_a, Rex_b or Rex_c (termed pMH-Rex, pMH-Rex_a, pMH-Rex_b and pMH-Rex_c, respectively) were generated by recombinant PCR to join exon 2 to exons 3, 3a, C and Ca, respectively. The resulting products were cloned in the expression vector pMHneo (Stratagene). HLtat cells were transfected with 1 µg of pMH-Rex, pMH-Rex_a, pMH-Rex_b or pMH-Rex_c. At 40 h after transfection, the cells were fixed in 4% formaldehyde (added to the culture medium) for 20 min, permeabilized with phosphate-buffered 0.1% NP40 for 10 min and treated with 100 µg/ml RNase for 1 h at room temperature. Cells were then incubated with a rabbit antibody (1:500) raised against amino acids 98–111 of Rex [16] for 1.5 h at 37°C, followed by incubation with an Alexa 488-conjugated anti-rabbit antibody (1:1,000, Molecular Probes) for 1 h at 37°C. Cells were then stained with 500 nM PI for 15 min at room temperature. Images were acquired with a Zeiss LSM 510 microscope using the Argon and Helium–Neon lasers (×63 optical magnification, ×4 scanning magnification). All parameters were standardized to allow comparison of signals obtained in different samples. Fluorescence signals were analyzed using a 505- to 530-nm band-pass filter for Alexa 488 and a long-pass 560-nm filter for PI.