Supplementary Figure 6: Immunostaining of DnaJA2 and other chaperones in samples from patients with MCI and AD.

Fixed brain sections from MCI, late-stage AD, or non-demented control (NDC) samples were co-stained for chaperone (red), phospho-tau (AT8, green) antibodies as well as the amyloid-binding small molecule, FSB (white). Nuclei were visualized with propidium iodide (PI) stain. a and b) Low magnification images show that MCI samples have a low incidence of AT8 positive neurons compared to late-stage AD samples. Scale bar = 100 microns. c) In an MCI sample, 3D rendering of one face of a neuron showing DnaJA2 is interspersed between or surrounds areas of AT8 staining. Colocalization between DnaJA2 and AT8 is not evident. The neuron shown is the same as presented in Fig. 5b (main text). d) Representative image from late-stage AD samples showing increased DnaJA2 staining adjacent (arrows), but not within, an AT8 and FSB positive neuron. Scale bar = 10 microns. e) DnaJA2 antibody does not detect other J-proteins such as the closely related family member, DnaJA1. Dot blot of recombinant chaperones with the same DnaJA2 antibody used for immunostaining. Ponceau stain of the same blot to verify protein transfer to membrane (representative of 2 independent experiments). f-h) Representative images of chaperone staining patterns in MCI and non-demented control samples. Scale bar = 10 microns for all images. Samples probed with antibodies against f) Hsp72, g) Hsp27 and h) Hsc70. Arrowheads mark neurons in h). Note that sections stained for Hsp27 could not be co-stained for phospho-tau (AT8).