Endogenous Human MDM2-C Is Highly Expressed in Human Cancers and Functions as a p53-Independent Growth Activator
Figure 5
MDM2-C increases with estrogen treatment in ER+ Breast cancer cells and is located in the cytoplasm and nucleus.
A. MCF-7 and T47D cells were grown and treated with 10nM estrogen (E2) for five days. Cells were lysed and proteins were analyzed via western blot. MDM2 C410 polyclonal serum anti-rabbit antibody was used for protein detection. Pre-immune polyclonal serum was used to detect background signal. HRP-conjugated anti-mouse and anti-rabbit were used as secondary antibodies. Actin was used as a loading control. This is representative of three independent experiments.
Bi. MCF-7 and T47D cells were lysed for whole cell or chromatin fractionation extracts. 50μg of samples were resolved using 10% SDS-PAGE and MDM2 C410 polyclonal sera (lanes 1 - 6) and MDM2 monoclonal antibody, 4B11 (lanes 13 - 18) were used for protein detection. Pre immune was used to detect background signal (lanes 7 - 12). Secondary antibodies used were same as in A.
Bii. Tubulin and Fibrillarin were used to determine fractionation purity.
C. Spinning disk microscopy of MCF-7 and T47D cells. Cells were grown on coverslips and treated with 10nM E2 for five days. Cells were fixed and incubated with p53 antibody and Mdm2 C410 polyclonal antibody for protein detection. Pre-immune serum was used as a negative control for staining. Slides were incubated with secondary Alexa-conjugated goat anti-rabbit and FITC-conjugated goat anti-mouse. DAPI was used to stain the nuclei. Cells were visualized at 60X magnification. Arrows depict MDM2-C localization regions.