It also represents a cellular model that mimics the early phases of the pathological state before aggresomes of R120G mutated HspB5 accumulate. Expression of wild type HspB5 often correlated with an elongated cellular morphology while expression of the R120G mutant induced the accumulation of membranous ruffles in the leading edges. These effects, which are probably linked to cytoskeleton reorganization, did not change the level of expression of stress markers, such as Hsps. R120G mutant expressing cells were also characterized by perinuclear granules, a phenomenon concomitant with the presence of almost 40% of the cellular content of both HspB1 and HspB5 in the particulate fraction upon cell lysis. Another dominant effect of HspB5 was observed at the level of the native size of HspB1. In presence of type HspB5, HspB1 oligomerization profile was lost and almost 90% of the total cellular amount of this protein interacted with HspB5. HspB5 R120G mutant acted similarly and interacted with HspB1 in a complex of slightly higher molecular mass. Analysis of the resistance to salt-mediated dissociation revealed that HspB1-HspB5 interaction was strengthened by the R120G mutation. This suggests that the interaction was more rigid and less dynamic. The other side of the coin concerned the protective effect of HspB1 towards HspB5. Previous reports mentioned that HspB1 could stabilize mutant HspB5 and attenuate the formation of inclusion bodies induced by expression of R120G HspB5. Using an RNAi approach, we confirmed that, in our cell system, HspB1 attenuates its ubiquitin-proteasome dependent clearance. We report here that cells expressing wild type HspB5 were more oxidoresistant than control cells while those expressing mutant HspB5 displayed a drastically enhanced sensitivity to oxidative stress. HspB5 molecular mechanism that drives oxidoresistance is not yet known. It could result of cytoskeletal protection, since this is a major cellular target rapidly altered in cells exposed to menadione, or modulation of intracellular redox status since HspB1 and HspB5 have been shown to decrease ROS levels and up-regulate the activity of different Lapatinib anti-oxidant enzymes, particularly G6PDH. Moreover, an effect towards HspB1, which gains in chaperone activity once it interacts with HspB5, LY2157299 cannot be excluded. On the other hand, the enhanced sensitivity mediated by the R120G mutation could be a direct consequence of the altered HspB1-HspB5 complex that cannot dissociate in a dynamic and controlled way and the lack of chaperone activity of mutant HspB5. This may lead to a defective recognition of oxidized polypeptides by these chaperones. Analysis of the phosphorylation status pointed to the increased phosphorylation of HspB1 and HspB5 in response to menadione treatment. This study also confirmed the already described hyperphosphorylation of mutant HspB5 in spite of the fact that this polypeptide is in a chimeric complex with HspB1. In that regard, it is interesting to note the R120G mediated enhanced phosphorylation was more intense for the serine sites that are close to the N-terminus; this could disturb hexamer contacts and favor HspB5 oligomer heterogeneity. Two-dimensional immunoblots analysis confirmed the acidic property of the isoforms suggesting that most R120G HspB5 molecules could be phosphorylated. Further analysis following fractionation of the different cell types revealed that the presence of these proteins in the pellet fraction correlated with their sensitivity to oxidative stress. Analysis of the phosphoserine sites of either HspB1 or HspB5 enlightened the different and complex patterns of phosphorylation of these polypeptides, particularly after oxidative stress, which drove a large fraction of these proteins in the particulate fraction. For example, it was first observed that, in the different untreated cells as well as in menadione-treated Neo.