FIG. 1.

Binding and effects of oral CHEC-9 in the frontal cerebral cortex of rats. (A) CHEC immunostaining in frontal cortex cytosol 150 min after 1 mg/kg CHEC-9 per os. Gels were run under non-reducing conditions. (Left panel) Immunopositive bands appeared only in CHEC-9–treated rats. Loading controls were partially transferred Coomassie Blue–stained gel bands (see Methods). (Right panel) Immunoprecipitation of cytosolic samples with CHEC-9 reactive antibody also comparing peptide- and vehicle-treated rats. Elution of the affinity matrix required boiling and chemical reduction of the samples prior to electrophoresis. These blots, immunostained for HSP70, showed bands also in the 70-kD region, prominent in CHEC-9–treated rats. Loading controls were glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Data are representative of five CHEC—vehicle pairs. (B) The response of cytosolic HSP70 to CHEC treatment. Multiple examples of paired CHEC-9– and vehicle-treated rats (C9 and V) run on 12.5% polyacrylamide gels. Individual pairs, shown before and after boiling and disulfide reduction, are aligned vertically. LC-loading control is GAPDH for reduced samples. (C) Two examples of cytosolic samples run on 7% gels in which higher-molecular-weight species containing HSP70 were recovered in vehicle-treated rats. (D) Quantification of the results. HSP70 immunostaining in the cytosol of rats treated with CHEC-9 was significantly increased in gels without heat denaturation and chemical reduction (unred.). After inclusion of these steps (red.), the differences were no longer significant. Plasma samples also show differences in HSP70 levels, in these cases independent of pretreatment (see text). n=8 for each condition. (**) p<0.01.