Cellular experiments
Cell culture, transient transfections, western-blot analysis, and immunocytochemistry were performed as described, [12-15] using antibodies against α-tubulin (Life Science) and acetylated α-tubulin (Sigma). Patients’ fibroblasts and adult control fibroblasts were grown under the same conditions, and analyzed among culture passages 5 and 8. As a positive control for tubulin acetylation, fibroblasts were pre-incubated with the specific HDAC6 inhibitor Tubacin (0, 0.2 μM and 2.5 μM) (Sigma) for 24 h [16]. Immunohistochemistry was carried out on 2 μm thick sections from pellets of Pt1, Pt2 and control fibroblasts, fixed in glutaraldehyde 2.5% (Electron Microscopy Science - EMS), in 0.05 M PBS pH 7.4, dehydrated in graded acetone, and embedded in Spurr (Epoxy resin, EMS).
Transfection of U251-MG by electroporation was performed in triplicate according to the manufacturer’s protocol (GenePulserII-Biorad), and about 100 cells were analyzed blindly for each experiment (a total of 324 cells for GFP-GFAP-ϵwt and 285 for GFP-GFAP-ϵR430H in a first experiment, and 460 cells for either GFP-GFAP-ϵwt or GFP-GFAP-ϵR430C in a second experiment).