Cloned wt PC12 and PC12-27 cells as well as the hChgB construct and the IgG2a anti-ChgB monoclonal were as in (Borgonovo et al. 2002); the IgG1 anti-hChgB monoclonal was the gift of W. Huttner, Dresden Germany; the anti-ChgA and anti-Scg2 polyclonals, of A. Laslop, Innsbruck Austria, and P. Rosa, Milan Italy, respectively; the 12C11-1 anti-REST monoclonal, of D. J. Anderson, Pasadena CA; the anti-ICA512 monoclonal, of M. Solimena, Dresden Germany; the goat and rabbit polyclonals against the Syt1 lumenal domain, of A. Malgaroli, Milan Italy; the MycREST and DBD/REST constructs, of N. Buckley, Leeds UK. Other antibodies were from commercial sources: anti-REST polyclonal, Upstate Cell Signaling, Lake Placid NY, USA; anti-Syt1, anti-Syp, anti-Rab3a, anti-VAMP2, anti-SNAP25 and anti-ß-tubulin monoclonals, Synaptic Systems, Göttingen, Germany; anti-Stx1a monoclonal, Sigma–Aldrich, St. Louis, MO, USA; anti-green fluorescent protein monoclonal, Roche, Indianapolis IN, USA; anti-Myc monoclonal, Abcam, Cambridge, UK; anti-GAPDH monoclonal, Biogenesis, Oxford, UK; FITC-conjugated and tetramethyl-rhodamine isothiocyanate-conjugated goat anti-mouse and anti-rabbit antibodies; goat anti-mouse IgG subclasses, Southern Biotech, Birmingham AL, USA; Alexafluor 647-conjugated anti-mouse antibodies, Molecular Probes, Eugene, OR, USA; horseradish peroxidase-conjugated goat anti-mouse and anti-rabbit antibodies, Bio-Rad, Hercules, CA, USA. trichostatin A (TSA) and cycloheximide were from Sigma-Aldrich; the BCA Protein Assay Kit from Pierce, Rockford, IL, USA.
Cell extractions; western blotting
Cell extracts in TD buffer containing 1% Triton X-100, 50 mM Tris–HCl (pH 7.5), 250 mM NaCl, 5 mM EDTA, 50 mM NaF and protease inhibitors, were rocked (15 min) and then centrifuged (13 000 g, 4°C, 15 min). Fixed amounts of protein, separated by SDS-PAGE and transferred to nitrocellulose filters, were processed as in Cocucci et al. (2004). Photographic development was by chemiluminescence (ECL Western Blotting Detection reagent of Amersham Biosci., Little Chalfont, UK or Femto Signal of Pierce). Signals were acquired by the Densitometer SI and Image Quant (Amersham Biosci.). Quantified western blot bands (averages of at least 3 experiments ± SD) were expressed as % of the corresponding values in wt PC12 cells.
Cells on coverslips fixed with 4% formaldehyde (10 min), quenched in 0.1 M glycine, washed, permeabilized in 0.1% Triton X-100 and immunolabeled as in Cocucci et al. (2004), were studied in Perkin-Elmer Ultraview ERS confocal microscope (Perkin-Elmer, Waltham, MA, USA). For surface immunolabeling the cells, stimulated or not for 1 min with 2 μM ionomycin, were fixed in 95% ethanol at 20°C, then washed and processed without detergent permeabilization to be finally labeled for Syt1 (lumenal domain), ChgB and ChgA, alone or in combination. Image deconvolution was carried out in a wide field microscope of the Delta Vision system (Cocucci et al. 2004). Fluorescence values were calculated on single cells by the ImageJ program (rsb.info.nih.gov/ij).
Monolayers of wt and PC12-27 clones and of stably transfected subclones were fixed with 2% glutaraldehyde in phosphate buffer, washed, post-fixed in 2% OsO4 in phosphate buffer and embedded in Epon. For immunogold labeling monolayers were fixed with a 4% formaldehyde/0.25% glutaraldehyde in phosphate buffer, washed, detached and recovered by centrifugation. Extensively washed pellets were embedded in London Resin white. Ultrathin sections on nickel grids were exposed to the anti-ChgB (90 min, phosphate-glycine buffer), washed and immunolabeled with gold particles (coated with anti-mouse IgGs, 6 or 10 nm), washed, post-fixed with 1% glutaraldehyde and finally stained in sequence with uranyl acetate and lead citrate. Ultrathin sections (60 nm) were studied and photographed in a Leo 912 electron microscope. The distribution, cytoplasmic density and radii of DCVs located away from the Golgi complex were measured manually on images chosen at random and printed at 50 000X. Correction of the radius measurements and calculations for volumes were made as in Parsons et al. (1995).