Permeabilized cells were incubated with 10% FCS in phosphate buffer and reacted with fluorescence tagged anti cytochrome c anti body. Cells were then washed and analyzed using flow cytometry. Data obtained were presented in concerning relative fluorescent units. Analysis of mitochondrial membrane potential To determine the perturbation of mitochondrial mem brane potential, the fluorescent cationic dye, iodide was used with FACS. The red to green ratio of JC 1 fluorescence is de tected to probe the m. When the integrity of mitochon drial membrane is maintained with high potential, JC 1 forms aggregates with red fluorescence. But the perturb ation of mitochondrial membrane leads it to emit only green fluorescence because of the loss of membrane po tential.
The mitochondrial membrane uncoupler, carbonyl cyanide m chlorophenylhydrazone, was used as a positive Inhibitors,Modulators,Libraries control that disturbs the mitochondrial mem brane potential. After loading the HeLa cells with JC 1 at 37 C, cells were analyzed by FACS using FL 1 and FL 2 channels. Identification of TCTP binding domain on Apaf 1 Several Apaf 1 deletion mutants that contain C terminal His tags were designed and constructed by referencing the previous study. Recombinant proteins including Full APAF 1, APAF 530, APAF 420, and APAF 97 were expressed in BL21 pLysS Escherichia coli and puri fied through affinity purification on a Ni Sepharose beads. WD Repeat of Apaf 1 protein was ob tained from Abnova Corporation. GST tagged recom binant TCTP protein was bacterially expressed in E. coli system and purified using GST fusion protein purifica tion kit.
Purified His Apaf Inhibitors,Modulators,Libraries 1 variants or WDR were immobilized on Handee spin column and then incubated with TCTP GST pro tein. Each eluates Inhibitors,Modulators,Libraries were separated by SDS PAGE and then subjected for silver staining. Results TCTP inhibits Inhibitors,Modulators,Libraries drug induced cell death by inhibiting the fragmentation of EGFR and PLC We attempted to clarify the antiapoptotic role of TCTP in the development of chemoresistance to etoposide, an inhibitor of topoisomerase II, as well as taxol, a microtubule stabilizer, those are widely used an ticancer drugs with distinctive modes of action. Human TCTP gene was overexpressed in human cervical cancer cells using adenoviral infection and apoptosis was measured by DNA fragmentation using propidium Inhibitors,Modulators,Libraries iodide staining and fluorescence activated cell sort ing.
We found that treatment with both etopo side and taxol increased cell death in HeLa cells. When TCTP was overexpressed, cell death decreased from 68% to 11%, in etoposide treated HeLa cells and from 71% to 13%, in taxol treated HeLa cells, confirming that TCTP inhibits cytotoxicity kinase inhibitor EPZ-5676 and cell death induced by two anticancer drugs. It has been suggested that etoposide as well as taxol cause apoptosis via caspase activation. Because EGFR and PLC are known to be cleaved by caspases during apoptotic process, we examined whether TCTP also inhibits the fragmentation of these proteins.