Figure 1.
Effect of N-Ethylmaleimide (NEM) on the distribution of endogenous caspase-2 (C2) (Panels A–C) and FLAG-ERα (Panel D) between nuclear and extra-nuclear (cytosol or lysate) cell fractions.
(A) Caspase-2 in cytosol and nuclei of cells without (−) or with (+) NEM (20 mM) pre-treatment for 10 min prior to cell fractionation using hypotonic buffer without detergent. (B) Caspase-2 in the lysate and nuclei of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. (C) Nuclear Caspase-2 in cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to lysis using the following conditions: 1) after lysis in cell culture plates (lysis buffer added directly to the plate wells), 2) cells in suspension collected in an Eppendorf tube were instantly frozen in dry ice/ethanol, transferred to ice bath and ice-cold lysis buffer with Triton X-100 was immediately added to the frozen pellets, 3) cells in suspension collected in an Eppendorf tube at room temperature and room temperature lysis buffer with 0.5% Triton X-100 was added to the pellets. (D). FLAG-tagged ERα in the lysate and nuclei of cells without (−) and with (+) NEM (20 mM) pre-treatment for 10 min prior to cell lysis with buffer containing 0.5% Triton X-100. The numbers on the right of the panels reflect the gel migration of the 40 kDa, 55 kDa, and 70 kDa protein markers. To ensure that our cell lysis procedure actually reflects nuclear and extra-nuclear (LYSATE) fractions, Western blotting studies examined for Histone H1.2 as a nuclear marker (E) and Procaspase-3 as an extra-nuclear marker (F) [31]. Cell lysis was carried out using the conditions given in (B) above.
Figure 2.
Effect of NEM and IAA concentrations on endogenous nuclear caspase-2 levels.
(A) Cells were pretreated with the indicated concentrations of NEM for 10 min before lysis. (B) Effect of NEM concentrations on nuclear caspase-2 levels. Cells were pre-treated with NEM as indicated for 1 h before lysis. (C) Effect of 10 min or 1 h pre-treatment with the indicated concentrations of Iodoacetic Acid (IAA) or NEM on caspase-2 levels in the nuclear fraction.
Figure 3.
Effect of leptomycin B on nuclear caspase-2 and FLAG-ERα levels.
(A) Cells were incubated with NEM (20 mM) for 10 min or with high levels of leptomycin B (100 nM) (LMB) for 2 h. Parallel-untreated cells (NONE) served as a control. The cells were then lysed and the level of endogenous nuclear caspase-2 determined by Western blotting. (B) Effect of zVDVAD-fmk on nuclear caspase-2 levels. Cells were incubated with a high level of zVDVAD-fmk (200 uM) for 15 h while parallel cells incubated with zVDVAD-fmk also received 20 mM NEM for 10 min prior to harvesting. Parallel-untreated cells (NONE) served as a control. The level of caspase-2 in the nuclear fraction was determined by Western blotting. (C) Similar to the study described in (A) except that the effect of leptomycin B and NEM was examined on nuclear FLAG-ERα levels 15 h after FLAG-ERα expression.
Figure 4.
Effect of NEM on the cell distribution of wild-type and mutant caspase- 2.
Shown is the distribution of wild-type GFP-Caspase-2 (GFP-C2-WT) (A), and mutant GFP-caspase-2 (GFP-C2-MUT) (B) between nuclear and extra-nuclear fractions of cells without (−) or with (+) NEM (7.5 mM) pre-treatment for 10 min prior to cell lysis. Also shown in both panels is the distribution of endogenously expressed caspase-2 in response to NEM pre-treatment in the same cells. The numbers on the right of the panels reflect the gel migration of the 40 kDa and 70 kDa protein markers.
Figure 5.
H2O2 incubation leads to rapid accumulation of caspase-2 in the extra-nuclear fraction of lysed cells.
(A) Caspase-2 in the extra-nuclear fraction (designated as lysate) after treating cells for 1 and 3 h with concentrations of H2O2 known to induce apoptosis. (B) Caspase-2 levels in the extra-nuclear fraction after treatment with 20 mM H2O2 for 5 or 15 min. After H2O2 incubation the medium was replaced with serum free medium containing 20 mM NEM for 10 min before lysis.
Figure 6.
Etoposide incubation leads to rapid accumulation of caspase-2 in the extra-nuclear fraction of lysed cells.
The results show the accumulation of caspase-2 in the extra-nuclear fraction (designated as lysate) after treating cells for 15 min to 3 h with 100 uM etoposide. After etoposide incubation the medium was replaced with serum free medium containing 20 mM NEM for 10 min before lysis.