However, the atx-3-null animals were significantly more resistant than wild type, with a median lifespan of 14 hours compared to 10 hours in wild type, corresponding to an increase of 40% in survival time in mutants . When we preexposed the animals to a 5-hour pre-heat shock PR-171 Proteasome inhibitor treatment, the median lifespans were 15 hours and 13 hours for atx-3 and wild type animals, respectively. These values corresponded to a 13% increase in survival time among mutants . When animals grown at 25uC were subjected to the same protocol, with the 2-hour pre-heat shock, atx-3 mutants�� median survival was 22 hours compared to 18 hours of N2 animals . In the case of the 5-hour pretreatment, the knockout animals�� median survival was 20 hours versus 17 hours for N2 animals . Real-time PCR results suggested that the atx-3 strain exhibited an enhanced activation of the chaperone machinery, at least at the mRNA level, during the heat shock. Our next step was to analyze the proteomic profile of atx-3 after a standard non-lethal heat shock in C. elegans. Detecting and quantifying whole proteins from a complex protein extract in a comprehensive manner remains a challenge in the NVP-BKM120 fields of proteomics, nevertheless, using the iTRAQ technique, which allows simultaneous quantification of 2�C8 samples by using different isotopes, we were able to obtain acceptable results. In the baseline condition, 35 proteins were altered in the atx-3 knockout animals when compared to wild type. These proteins belong to several heterogeneous classes such as ribosomal proteins, vitelogenins and histones. After heat shock, 148 proteins were significantly altered; with a predominance of ribosomal proteins, molecular chaperones, enzymes and histones. The most consistent difference was in the expression of HSP-16 family members – HSP-16.1 and HSP-16.49, which were clearly up-regulated in atx-3 strains. We were unable to quantify the levels of HSP-16.2 protein. The SIP-1 protein was altered in the knockout strains but displayed a divergent profile in the biological replicates we analyzed. Other chaperones such as HSP-3, -6, -12.2 and -60 were present at similar levels in wild type and atx-3 mutant animals while HSP-12.6 levels were diminished. We aimed to confirm some of these findings by western blot. Aside from analyzing the time-course of the heat shock, as previously performed using real-time PCR, we also analyzed the chaperone profile of the recovery after heat shock. We were able to confirm HSP-16 overexpression using anti-HSP16 antibody. Since this antibody recognizes several members of the HSP-16 family, the overexpression detected was less pronounced than in the proteomic analysis, probably due to the masking effect of other proteins of this family, which were unaltered in the atx-3 animals. We found that HSP-16 proteins were first detected 60 minutes after the beginning of heat shock. HSP-16 levels were elevated in atx-3 animals when compared to controls at 60�C90 minutes of heat shock.