Figure 1.
The protein-protein interactions of BIFC constructs in living cells.
(A) BiFC detected by flow cytometry of Cos-1 cells transfected with empty vectors, pBiFC-VN173 and pBiFC-VC155 by varying amounts of DNAs (125–1000 ng) at each time point as indicated. (B) Confocal image of Cos-1 cells gave no detectable Venus fluorescence when 250 ng of DNA for control vectors were used after 12h transfection. (C–E) Fluorescence images of Cos-1 cells expressing the VC and VN pairs indicated in each panel under the same conditions. The strong BIFC signal was monitored by confocal microscopy in hNUDC-VC/Mpl-VN- or hNUDC(1-159)-VC/Mpl-VN-transfected cells, but no fluorescence was seen in cells co-transfected with hNUDC(160-331)-VC and Mpl-VN. The cells were stained with DAPI to indicate the position of cell nucleus (Original magnification, 40x).
Figure 2.
The BiFC was quantified by flow cytometric analysis and Western blot.
Cos-1 cells were transfected with pairs of BiFC constructs or with control constructs as indicated for 12 h. For each combination, the mean fluorescent intensity of YFP from 1×104 cells was determined. (A) Dot plots showing the R2 gate delimiting the area of BIFC signals. (B) The relative levels of the specific fluorescence signals are shown as a histogram. Data are representative of at least 4 independent experiments. *, p<0.05; **, p<0.01 compared to untransfected Cos-1 cells. (C) Examination of expression levels of BiFC constructs by Western blot. Cos-1 cells co-expressing the indicated BiFC constructs were lysed and incubated with anti-HA or Flag antibodies. Locations of molecular weight markers are indicated.
Figure 3.
Cell images from standard acceptor photobleaching FRET.
(A–C) Cos-1 cells transfected with plasmids encoding the CFP-YFP fusion proteins as indicated. (D) Representative cells transfected with empty vectors as a negative control for FRET. The white boxes indicated CFP and YFP images before and after photobleaching by a high intensity argon laser light. Five ROIs were analyzed for each cell and at least 5 cells were quantified from three different experiments. (E) FRET efficiencies calculated with Leica LAS AF software during YFP photobleaching. All data are represented as mean±S.D. *, P<0.05 compared to untransfected Cos-1 cells.
Figure 4.
Subcellular localization of BiFC constructs.
(A) Representative BiFC distribution of hNUDC-VC/Mpl-VN is shown. (B) Representative BiFC distribution of hNUDC(1-159)-VC/Mpl-VN is shown. BiFC constructs were co-transfected with the indicated subcellular markers. Fluorescent images shown were captured 12 h after transfection. Merged images show good co-localization of hNUDC/Mpl or hNUDC(1-159)/Mpl with the ER, Golgi and cell membrane. The cells were stained with DAPI to indicate the position of cell nucleus. Arrows, regions of co-localization; scale bar, 5 µm.
Figure 5.
Western blot analysis of hNUDC domains in the medium of cultured Dami cells.
(A) Dami cells were transfected with Ad-hNUDC. (B) Dami cells were transfected with Ad-hNUDC(1-159). (C) Dami cells were transfected with Ad-hNUDC(160-331). Secreted hNUDC, hNUDC(1-159) or hNUDC(160-331) in the culture medium was assayed by immunoblot using an anti-hNUDC antibody at each time point indicated.
Figure 6.
Western blot analysis of hNUDC domains in the medium of cultured from Mpl knockdown Dami.
(A) Effects of siRNA-mediated Mpl knockdown. Dami cells were transfected with Ad-ShRNA-Mpl. Total cell proteins were prepared at the indicated times, and the effects of Ad-ShRNA-Mpl transfection on Mpl expression levels were evaluated by immunoblotting using an anti-Mpl antibody. Determination of the protein levels of β-Actin (bottom) was used as a control to ensure equal protein loading. (B) Time course of hNUDC released into culture medium after Dami cells were co-transfected with Ad-hNUDC and Ad-ShRNA-Mpl. (C) Comparison of hNUDC released into culture medium in cells transfected with Ad-hNUDC and control Ad-shRNA-scramble. (D) Time course of hNUDC released into culture medium after Dami cells were co-transfected with Ad-hNUDC(1-159) and Ad-ShRNA-Mpl. (E) Comparison of hNUDC(1-159) release into culture medium in Dami cells transfected with Ad-hNUDC(1-159) and control Ad-shRNA-scramble. Proteins released into the culture medium were analyzed by immunoblot using an anti-hNUDC antibody at each time point as indicated. Results are from 3–5 independent experiments.