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DjB1 (Hsp40/DnajB1/Hdj1) is a member of the Hsp40/DnaJ family that functions as a co-chaperone of mammalian Hsp70s. DjB1 recognizes substrate proteins and facilitates the ATPase activity of Hsp70. We generated DjB1 deficient mice. The DjB1–/– mice were viable and fertile with no obvious abnormalities, thus indicating that DjB1 is dispensable for development and viability. No difference was found between the DjB1–/– and wild-type peritoneal macrophages regarding resistance against various types of apoptosis-inducing reagents. However, DjB1–/– cells showed decreased thermotolerance in the early phase after mild heat treatment, but not in the late phase. After the heat treatment, Hsp70 was induced similarly in wild-type and DjB1–/– cells. Immunofluorescence staining of wild-type cells revealed the accumulation of DjB1 and Hsc70 in the nucleus after heat treatment. DjB1 also accumulated in the centrosome. The accumulation of Hsc70 in the nucleus was also observed in DjB1–/– cells. These results suggest that the impaired thermotolerance of DjB1–/– cells is not due to a mislocation of the Hsp70 family. [ABSTRACT FROM PUBLISHER]