Ix81 fv1000 d fluoview system

HeLa cells (4 × 10⁴) were seeded overnight on cover glass in 12-well plates before transfection for 48 h with 0.8 μg of pCAGGS/Gag and 1.5 μg of pCAGGS/eCFP-TSG101 without/with 0.5 μg of pCAGGS/HA-Vpr, 0.5 μg of pCAGGS/HA-Vpr A30F, or 0.5 μg of pCAGGS/Vif-HA plasmids. After 48 h transfection, the cells were processed to immunofluorescent stain as described previously [31 (link)] with the following antibodies: anti-Gag rabbit polyclonal antibody (pAb) (Bio Academia), anti-HA mouse monoclonal antibody (mAb) (Medical and Biological Laboratories Co., LTD), anti-LAMP1 mouse mAb (Santa Cruz Biotechnology), anti-EEA1 rabbit mAb (Cell Signaling), anti-Rab7 rabbit mAb (Cell Signaling), anti-Rab11 rabbit pAb (Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen), and Alexa Fluor 633 goat anti-mouse IgG (Invitrogen). Image acquisition was performed at the followed excitation/emission wavelengths (Ex./Em.): Alexa Fluor 594 at 543/618 nm, Alexa Fluor 633 at 635/647 nm, and eCFP at 405/476 nm, under a confocal laser-scanning microscope (IX81-FV1000-D/FLUOVIEW System, Olympus). Gag/TSG101 co-localization was analyzed by Pearson’s correlation coefficients (linear regression of Gag and TSG101 fluorescent intensity plot which 1 is a total positive correlation, 0 is no correlation, and −1 is a negative correlation) using FV10-ASW v.2.1 software (Olympus).

HeLa cells (2.5 × 10⁵) or HEK293 cells (2.5 × 10⁵) were seeded on cover glasses in a 12-well plate and transfected with 1 μg of pcDNA 3.1/HA-MCM10, with or without 1 μg of pcDNA3.1/3 × FLAG-Vpr. Following 48 h of transfection, immunofluorescence staining was performed as described previously [24 (link)]. In brief, cells on a cover slip were fixed with 4% paraformaldehyde for 10 min at room temperature. Paraformaldehyde was then replaced with cold methanol and the cells were maintained at −20 °C for 20 min. The cells were then washed with PBS and incubated with anti-FLAG rabbit mAb (MBL), anti-HA mouse mAb (MBL), or anti-gamma H2AX mouse mAb (Abcam, Cambridge, UK) for 1 h at room temperature. Following further washing with PBS, Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, Waltham, MA, USA) or Alexa Fluor 594 goat anti-mouse IgG (Invitrogen) was added for 1 h at room temperature in the dark. Nucleus was stained with Hoechst 33342 (Thermo Fisher Scientific) for 5 min in the dark. Coverslips were then rinsed with PBS and mounted on glass slides. Processed samples were visualized using a confocal fluorescence microscope (IX81-FV1000-D/FLUOVIEW System, Olympus, Tokyo, Japan).

HeLa cells (4 × 10⁴) were transfected with 0.8 μg of pCAGGS/Gag-Venus (or pCAGGS/Venus only) or with 1.5 μg of pCAGGS/eCFP-TSG101 (or pCAGGS/eCFP only) without/with 0.5 μg of pCAGGS/mRFP-Vpr plasmids for 48 h, fixed with 4% paraformaldehyde, and then visualized using a IX81-FV1000-D/FLUOVIEW System (Olympus). The fluorescent signal generated by the eCFP donor was acquired at an Ex. of 405 nm (Ex.eCFP) and an Em. of 460–500 nm (Em.eCFP). The fluorescent signal generated by the Venus acceptor was acquired at Ex. of 515 nm (Ex.Venus) and Em. of 515–615 nm (Em.Venus). The uncorrected FRET signal was acquired at the Ex.eCFP and Em.Venus. The precision FRET (PFRET) signal was generated using the sensitized emission method by subtracting the uncorrected FRET signal from the signal generated by spectral bleed-through into the acceptor channel (the fluorescent signal from the donor emitted into the acceptor channel and the fluorescent signal generated by excitation of the acceptor at the donor excitation wavelength), which is derived from eCFP-TSG101 only and Gag-Venus only samples [32 (link)]. This process was automatically calculated by FV10-ASW v.2.1 software (Olympus). The FRET ratio was calculated by dividing the PFRET signal with the donor eCFP-TSG101 signal.