Exogenous protein Hsp70/Hsc70 can penetrate into brain structures and attenuate the severity of chemically-induced seizures
Article first published online: 7 OCT 2010
© 2010 The Authors. Journal of Neurochemistry © 2010 International Society for Neurochemistry
Journal of Neurochemistry
Special Issue: Introducing Preclinical Systematic Reviews
Volume 115, Issue 4, pages 1035–1044, November 2010
How to Cite
Ekimova, I. V., Nitsinskaya, L. E., Romanova, I. V., Pastukhov, Y. F., Margulis, B. A. and Guzhova, I. V. (2010), Exogenous protein Hsp70/Hsc70 can penetrate into brain structures and attenuate the severity of chemically-induced seizures. Journal of Neurochemistry, 115: 1035–1044. doi: 10.1111/j.1471-4159.2010.06989.x
- Issue published online: 21 OCT 2010
- Article first published online: 7 OCT 2010
- Accepted manuscript online: 10 SEP 2010 05:55PM EST
- Received June 9, 2010; revised manuscript received August 31, 2010; accepted September 2, 2010.
Figure S1. Characteristics of the Hsp70/Hsc70 preparation isolated from red bovine muscle in comparison with recombinant human Hsp70. (a) Electrophoretic profile of both preparations, bovine Hsp70/Hsc70 and human recombinant Hsp70, and western blotting analyses with anti-Hsp70 (Clone 116) and anti-Hsc70 (Clone N69, Novoselova et al., 2005) monoclonal antibodies. For Coomassie staining of the gel 2 μg of each preparation was used, for western analysis 10 ng was used. (b) Analysis of protein-binding (‘chaperonic’) activity of bovine Hsp70/Hsc70 and recombinant hHsp70 was determined using the ELISA method (Wawrzynow and Zylicz 1995), see Materials and methods. (c) Analysis of the protective activity of bovine Hsp70/Hsc70 and recombinant hHsp70. Human neuroblastoma SK-N-SH cells were incubated with 500 nM staurosporine alone or in combination with 5 or 25 μg/mL of bovine Hsp70/Hsc70 or recombinant hHsp70 preparation. The level of apoptosis was estimated as the percentage of cells with fragmented nuclei stained by Acridine orange.
Figure S2. Effects of Hsp70/Hsc70 on epileptiform brain activity and contractile muscle activity during seizures induced by NMDA. (a) Encephalogram (EEG) recordings from the motor cortex of vehicle-treated, NMDA-treated and Hsp70/Hsc70-treated animals; note the NMDA-induced high amplitude spike-wave discharges and their reduction in the case of Hsp70/Hsc70-NMDA treatment. (b) Number of EEG spike-wave complexes. (c) Duration of EEG spike-wave complexes. (d) The level of neck muscle contractile activity of NMDA-treated and Hsp70/Hsc70 + NMDA-treated animals. Three or six micrograms of Hsp70/Hsc70 was injected into the third ventricle of the brain as described in the Material and methods in advance of NMDA administration and the effects of both factors were determined as described in the text.
Figure S3. Microinjected Hsp70/Hsc70 crosses the cerebrospinal fluid-brain barrier and is not internalized by brain microglia. Six micrograms of Bodipy ® FL-labelled Hsp70/Hsc70 was injected into the third ventricle of the brain as described in the Materials and methods. Two hours later rats were killed and brain slides were prepared and stained with specific anti-CD11b antibody (red). Hsp70/Hsc70 localization is seen as green spots. (a) Image obtained using light microscopy. (b) Images obtained using confocal microscopy. See Materials and methods for details. Scale bar is 10 μm.
Figure S4. Fluorescently-labelled Hsp70/Hsc70 migrates to NMDA receptor-rich regions of the brain. Rat brain slides were stained with an antibody recognizing the post-synaptic marker protein NMDAR1 (NMDA glutamate receptor subunit R1) (red). Scale bar is 10 μm.
Figure S5. Characteristics of monoclonal anti-Hsp70 antibody (Clone 116). Human erythroleukemia K-562 cells and rat rhabdomyosarcoma RA-2 cells (Guzhova at al. 1992) were subjected to heat shock (43°C, 60 min) and allowed to recover for 6 h. Control and heat shocked cells were collected, lysed and 50 μg of each sample was used for western blot analysis. One part of the membrane was probed with 3C5 monoclonal antibody recognizing the epitope at the N-terminal part of the Hsp70 molecule (Lasunskaia et al. 2010), and another with the 116 monoclonal anti-Hsp70 antibody. The antibody to Hsc70 (N69) was used for loading control.
Figure S6. Both members of the Hsp70 family, inducible Hsp70 and constitutive Hsc70, were found on brain slices. Slices of the anterior hypothalamus of the rat after microinjection (i.c.v.) of Bodipy ® FL labelled Hsp70/Hsc70 preparation (2 h) were probed with monoclonal anti-Hsp70 (Clone 116), upper panel, or anti-Hsc70 (Clone N69), lower panel, followed by Cy3-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories INC, 115-165-146, USA). Scale bar is 10 μm.
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