Z0458

Early trophozoite stage P. falciparum-infected red blood cells (21–24 h post invasion, 5% haematocrit) were incubated with 1 μM DHA and 400 nM epoxomicin or with 400 nM epoxomicin alone in the presence of 0.1% DMSO (carrier solvent), for 3 h at 37 °C. Treated parasite cultures were attached to erythroagglutinin PHA-E-coated glass slides, incubated in 50 μM TPE-MI for 30 min at 37 °C and fixed with 2% formaldehyde and 0.008% glutaraldehyde before imaging.
For the western blot experiment, red blood cells infected with trophozoite stage P. falciparum at 5% parasitemia and 5% haematocrit, were exposed to vehicle (0.1% v/v DMSO) or 400 nM epoxomicin and/or 1 µM DHA for 3 h. Parasites were harvested for analysis of ubiquitinated proteins as previously described^{47 (link)}. The antibodies used were rabbit anti-ubiquitin (Dako-Z0458) and goat anti-rabbit IgG-peroxidase (Sigma-Aldrich-A0545), with dilutions 1:100 and 1:25,000, respectively.

Mice were deeply anaesthetized and trans-cardially perfused with 4% PFA. After removal of the spinal cord, spinal cord was post-fixed in 4% PFA overnight and subsequently incubated in 30% sucrose at 4 °C overnight. Thirty-five micrometers of thick free-floating sections were cut on a Leica 9000 s sliding microtome as described previously^{62 (link)} and collected in 0.1 M phosphate buffer (PB) pH 7.4. After incubation of the sections for 1 h with 4% normal goat or donkey serum and 0.3% Triton X-100 for blocking of nonspecific binding at room temperature, sections were incubated overnight at 4 °C with primary antibodies in blocking solution. After three times 10 min washing in 0.25% Triton X-100 in PB at room temperature, sections were incubated in with fluorescently labeled secondary antibodies, washed again and finally mounted with Mowiol/DABCO. The following primary antibodies were used: anti-NeuN (1:1000; Millipore, MAB377, clone A60), ChAT (1:1000; Millipore, MAB144P), anti-Ubiquitin (1:500; DAKO, Z0458), anti-Synaptophysin-1 (1:500; Synaptic Systems, 101 004). Alexa-647-, Alexa-488-, and Alexa-546-conjugated secondary antibodies were from Jackson Immuno-Research Laboratories. For visualization of F-actin Alexa Flour 532 conjugated Phalloidin (Invitrogen) was used.

Cultures were fixed in freshly prepared, buffered 4% paraformaldehyde. After blocking with 10% normal goat serum (NGS), cultures were incubated overnight at 4 °C with the following antibodies (mAbs, monoclonal; pAbs, polyclonal): β-tubulin isotypeIII (β-tubIII, mAbs, MMS-435 P (Covance, Princeton, NJ, USA), 1 : 400), GFAP (pAbs (Dako, Cernusco sul Naviglio, Milan, Italy), 1 : 400), Lamp1 (pAbs, ab24170 (Abcam, Cambridge, UK), 1 : 750), cleaved caspase-3 (Casp; Asp 170) (pAbs, no. 9961 (Cell Signaling Technology, Danvers MA, USA), 1 : 500), Ub (pAbs, Z0458 (Dako), 1 : 50) MAP2 (mAbs (Chemicon, EMD Millipore, Darmstadt, Germany), 1 : 400).
After removal of the primary Abs and repeated washes with Dulbecco's phosphate-buffered saline (PBS), cultures were incubated at room temperature for 45 min with secondary antibodies labeled with Alexa Fluor 594 or 488 (anti-mouse and/or anti rabbit; Molecular Probes, ThermoFisher Scientific, Waltham, MA, USA). Samples were then colored with DAPI (4′,6-diamidino-2-phenylindole; 0.3 μg/ml; Roche, Basel, Switzerland) for nuclear staining and rinsed with PBS for mounting and analysis. Microphotographs were taken using a Zeiss Axiovert 200 direct epifluorescence microscope (Axioplan 2; Carl Zeiss, Jena, Germany) or a confocal microscopy (Leica DM IRE2, Milan, Italy).

At autopsy, brain, spinal cord and other organs were fixed in 10% formalin solution. Samples of the main representative regions of each organ were embedded in paraffin and sectioned in 6 -μm thickness, and stained with haematoxylin and eosin and Klüver-Barrera. Skin biopsy, and following immunostaining and immunofluorescence staining were performed as described previously (Sone et al., 2011 (link)) with anti-ubiquitin antibody (Z0458, Dako) and anti-p62 antibody (sc-28359, Santa Cruz Biotechnology). For assessment of intranuclear inclusion frequency, we prepared a coronal section of cerebral hemisphere, including basal ganglia, stained with anti-p62 antibody using the LSAB Kit (Dako). We selected five random microscopic fields under a ×20 objective lens in each gyrus or basal ganglion, counted the number of neurons, astrocytes and p62-positive intranuclear inclusions and assessed the frequency of intranuclear positive neurons and astrocytes. Samples for electron microscopic study were prepared as described previously (Koike et al., 2007 (link)).

Equal volumes of the detergent-insoluble fractions were separated on 4–15 % Criterion® TGX gels (Bio-Rad) and analyzed using western blot with anti-ubiquitin antibody (1:25,000; Z0458; Dako), which reacts with both Lys48- and Lys63-linked chains [50 (link)]. β-Actin in whole homogenates (1:50,000; MAB1501R; Millipore) was used as an internal marker.