Figure 4
From: Blocking nuclear export of HSPA8 after heat shock stress severely alters cell survival
Cross-linking experiments to locate the binding site of P140 on HSPA8. (A) Western immunoblotting analysis showing HSPA8 protein after photo cross-linking experiments with Atf-biotin-P140 peptide (UV irradiation time is indicated in min). HSPA8 was transferred from denaturing gel to polyvinylidene difluoride membranes and stained with HRP-conjugated streptavidin, and developed with electrogenerated chemiluminescence detection reagent. (B) Positive reflectron MALDI-TOF spectra of HSPA8 NBD standardized as described in the materials and methods section. Trypsic digestion of NBD-containing fragment was monitored in the presence (right panel) or absence (left panel) of the cross-linking (one representative of three independent experiments with three spots/experiment). The arrows indicate the position of two peaks that vary in the cross-linked versus non-treated protein. (C) Sequence, mass and MALDI-TOF signal intensity ratios of each peptide generated in the presence or absence of cross-linking. The SD values were calculated from the 9 ratios (3 independent experiments with three spots/experiment). The ratios were considered as significant (red) if they were higher than a 3-fold change. (D) Left: depiction of the space-filling model of HSPA8 NBD showing the ATP pocket (blue) and the BAG-1-interacting domain (yellow). Right: Ribbon illustration of HSPA8 NBD with the amino acid sequences of interest highlighted. The location of NLS (green) and NoLS (cyan) motifs are highlighted (PDB code access 3HSC). The peptides 88–101 and 272–298, whose detection by MALDI-TOF experiments is influenced by the presence of cross-linking, are shown in fuchsia pink in both panels.
