is modification affects Hax 1 stability will be of help to explore the exact role of the PEST sequence in Hax 1. Hax 1 is structurally similar to Bcl 2 for its BH domains and TM domain. However, Hax 1 is less stable compared to other Bcl 2 family proteins. It was reported that Hax 1 is rapidly cleaved by caspase 3, HtrA2 or Granzyme B during cell death. It is therefore possible that these enzymes contribute to Hax 1 degradation in apoptosis. As Hax 1 is a short lived protein and also degraded by the Entinostat proteasome, it suggests that the proteasomal degradation of Hax 1 highly regulates Hax 1 levels in normal conditions. Knockdown of pleiotropic human prohibitin 2 in HeLa cells results in caspase dependent apoptosis through down regulation of Hax 1.
Here, we report that, in addition to protease cleavage, the proteasomal degrad ation is also an important post translational regulation for Hax 1 during apoptosis. When the PEST sequence is abolished, Hax 1 is shown to con vey increased resistance to cell death. Taken together, these data suggest that Hax 1 may be rapidly subjected to proteolysis in response to cellular stresses, resulting in a decrease in its protein level and hence loss of its protective activity. Conclusions In summary, our study demonstrates that Hax 1 is rapidly degraded by the proteasome in a PEST se quence dependent manner. During apoptosis, degrad ation of Hax 1 is enhanced whereas expression of PEST mutant of Hax 1 protects cells against apop totic stimulation.
Methods Cell culture, transfections and drug treatments N2a and H1299 cells were grown in Dulbeccos Modi fied Eagles Medium containing 10 % fetal calf serum with 100 ug ml penicillin and 100 ug ml streptomycin. Transfections were performed using Lipofectamine 2000 according to the manufacturers instructions. In order to ensure equal transfection efficiency, master mix of the same plas mids were made and aliquot to each well, we double check the equal expression of EGFP Hax 1 through fluoresce microscopy before drug treatment. Hoechst 33342, DAPI, STS, Bafilomycin A1, Annexin V, PI and CHX were purchased from Sigma. MG132 was obtained from Calbiochem. 35 pmoles of each siRNA were transfected using Oligo fectamine, according to the manufacturers instructions. Oligonucleotides were purchased from GenePharma and had the following sequences, Immunoblot analysis and antibodies Cell extracts were lysed in 1 �� RIPA lysis buffer in the presence of pro tease inhibitor cocktail.
Approximately 20 ug of cell lysates was separated on SDS PAGE and trans ferred onto a PVDF membrane. Immuno blot analyses were carried out with the following primary antibodies, anti Bcl 2, anti Bcl xL, anti GAPDH, anti GFP, anti LC3, anti Tubulin, anti Hax 1, anti Flag, anti ubiquitin, anti K48 ubiquitin and anti K63 ubiquitin. The second ary antibodies, i. e. sheep anti mouse IgG HRP or anti rabbit IgG HRP, were from Amersham Pharmacia Biotech. The proteins were visualized using an ECL detection kit. Immunopr