Dr. K.Y. Chen's Laboratory - Hypusine and eIF5A

Subcellular Localization of eIF5A

Subcellular localization of eIF5A by indirect immunofluorescence with two different fixation protocols.

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Subcellular localization of eIF-5A by indirect immunofluorescence with two different fixation protocols.

Immunofluorescence(left-hand panel) and corresponding phasecontrastimages (right-hand panel) of COS-7 cells fixed with4% paraformaldehyde (A and B; E and F) or methanol (C and D)are shown. Fixed cells were treated with the purified anti-eIF-5Aantibody (1:20). FITC-conjugated rabbit anti-chicken immunoglobulin(IgG) was used as the secondary antibody to visualizethe protein. As a control, fixed cells were also probed with theanti-eIF-5A antibody that was pre-absorbed with recombinant human eIF-5A proteins (E and F).

Bar: 25 μm.

Expression and localization of GFP-eIF5A in transiently transfected COS-7 cells.

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Expression and localization of GFP-eIF-5A in transiently transfected COS-7 cells.

(A) Western blot analysis of GFPeIF-5A in the transfected cells.

(B) Direct visualization of GFPeIF-5A.

(C) Indirect immunofluorescent staining of GFP-eIF-5A
in the transiently transfected cells (fixed with 4% paraformaldehyde).

(D) Indirect immunofluorescent staining of the same cells
in methanol.

Bar: 25 μm.

Staining of eIF5A and calnexin or eIF5A and CRM1 by double immunofluorescence microscopy.

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Staining of eIF-5A and calnexin (top panels) or eIF-5A and CRM1 (bottom panels) by double immunofluorescence microscopy. COS-7 cells were fixed with formaldehyde or methanol and then stained with anti-eIF-5A and anti-calnexin antibodies (top panel) or anti-eIF-5A and anti-CRM1 antibodies(bottom panel). FITC-fluorescence images revealed the distribution of eIF-5A (A, E, I, and M). Cy3-fluorescence images revealed the distribution of calnexin (B and F) or CRM1 (J andN). Double immunofluorescence images revealed the mergedimages of eIF-5A and calnexin (C and G) or eIF-5A and CRM1(K and O). Phase contrast images of the corresponding fixedcells are shown in (D), (H), (L), and (P). Bar: 25 mm.

Expression and localization of GPK-eIF5A in transiently transfected COS-7 cells.

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Expression and subcellular localization of GPK-tagged eIF-5A domain chimera proteins in living cells.

(A) Western blotanalysis of various GPK-eIF-5A fusion proteins using anti-eIF-5A or anti-GFP antibody. Note that GPK-eIF-5A(183) could not be detected by the anti-eIF-5A antibody, since the epitopes of this antibody are located to the C-terminal portion of eIF-5A.  However, it can be detected by anti-GFP antibody.

(B) Direct visualization of GPK-eIF-5A (aa l83).

(C) Direct visualization of GPK-eIF-5A (aa 41120).

(D) Direct visualization of GPK-eIF-5A (aa 84154).

(E) Direct visualization of GPK-eIF-5A (KSOR).

Expression and subcellular localization of GPK-tagged eIF-5A domain chimera proteins in living cells.

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Expression and subcellular localization of GPK-tagged eIF-5A domain chimera proteins in living cells.

(A) Western blotanalysis of various GPK-eIF-5A fusion proteins using anti-eIF-5A or anti-GFP antibody. Note that GPK-eIF-5A(183) could not be detected by the anti-eIF-5A antibody, since the epitopes of this antibody are located to the C-terminal portion of eIF-5A.  However, it can be detected by anti-GFP antibody.

(B) Direct visualization of GPK-eIF-5A (aa l83).

(C) Direct visualization of GPK-eIF-5A (aa 41120).

(D) Direct visualization of GPK-eIF-5A (aa 84154).

(E) Direct visualization of GPK-eIF-5A (KSOR)

Heterokaryon analysis of the nuclear export of eIF5A

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EHeterokaryon analysis of the nuclear export of eIF-5A. HeLa cells were transfected with GPK-Rev-NLS, GPK-M10-NLS, or GPK-5A-NLS and then fused with mouse 3T3 cells as described under Materials and Methods. (A), (D), and (G) are the intrinsic GFP signals of GPK-Rev-NLS, GPK-M10-NLS, and GPK-5ANLS, respectively. (B), (E), and (H) are the PI staining of the same cells in (A), (D), and (G), respectively. (C), (F), and (I) are the phase-contrast images in (A), (D), and (G), respectively. Arrows denote the mouse nuclei.

Bar: 10 μm.

Effect of detergent extraction on the immunostaining patterns of eIF5A and GFP in the COS-7 cells.

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Effect of detergent extraction on the immunostaining patterns of eIF-5A and GFP in the COS-7 cells.

COS-7 cells were treated with 0.2% Triton X-100 before fixation as described under Materials and Methods.

(A) Immunofluorescent staining of the Triton-extracted COS-7 cells using anti-eIF-5A antibody.

(B) Phase-contrast image in (A).

(C) Immunofluorescent staining of the Triton-extracted GFP-transfected COS-7 cells using anti-GFP antibody.

(D) Phase-contrast image in (C).

Bar: 25 μm.