Figure 3. Western blotting in conjunction with immunoprecipitation identifies increased cytoplasmic Cyt c in lrg1-transfected cells and, following degradation of LRG1 in apoptotic cells, binding of Cyt c to Apaf-1.

(a) Antibodies specific for COX IV, a mitochondrial marker, confirmed by Western blotting the purity of the subcellular fractions of MCF-7 cells expressing different amounts of LRG1. LRG1 was detected in the cytosol (S-100 fraction) of lrg-transfected MCF-7 cells and to a lesser extent in the cytosol of parental cells using a more sensitive detection reagent. Cyt c was also found in the cytosol of live lrg1-transfected cells, although the amount of Cyt c-co-immunoprecipitated with Apaf-1 was weakly detected. Asterisks indicate the use of a more sensitive detecting reagent.

(b) LRG1 was degraded in peroxide-treated cells, however, degradation was not blocked by the proteasome inhibitor bortezomib (btz). Cyt c in the cytosol of lrg1-transfected cells was co-immunoprecipitated with recombinant LRG1. Less Cyt c was present in the pull down from cells treated with hydrogen peroxide, consistent with degradation of LRG1.

(c) In the cytosol of apoptotic cells, the amount of Cyt c co-immunoprecipitated with Apaf-1 was inversely related to the amount of LRG1 expressed. Results for two independent trials are shown.

(d) Degradation of LRG1 in hydrogen-peroxide-treated parental MCF-7 cells was confirmed by confocal microscopy. Cells were labeled using mAb 3C9.D5 specific for LRG1 and nuclei were stained using DAPI. Live cells are on the left and cells treated for 3 hrs. with 0.1% hydrogen peroxide are on the right.