Skip to main content
. 2011 Sep 30;201(1):228–238. doi: 10.1016/j.jneumeth.2011.08.003

Fig. 1.

Fig. 1

Western blots showing that active chondroitinase is secreted following transduction of tissue culture cells with the lentiviral vectors. Neu7 conditioned medium (a source of CSPGs) was placed on transfected cells for 24 h, then analyzed by western blotting. (a) LV-C and LV-D. The first two lanes are positive and negative controls with Neu7 medium not exposed to transfected cells; lane 1 was digested in vitro with commercial chondroitinase (Sigma, 20 mU, 37°, 3 h). (Top panel) Probed for carbohydrate ‘stub’ epitope produced by chondroitinase action (antibody 1B5). Neu7 conditioned medium (lane 2) shows little immunoreactivity, but incubation with cells after transduction with LV-ChABCs generates extensive reactivity. (Middle) Probed for NG2. Undigested NG2 (lane 2) appears largely as a characteristic ‘smear’ above the core protein band due to the heterodispersed high-Mr GAG chains, and this is all converted to core protein by digestion with commercial chondroitinase (lane 1) or by incubation with cells after transduction with LV-ChABC. (Bottom) Probed for chondroitinase ABC. Commercial chondroitinase (lane 1) shows both full-length band (Ch) and a shorter band (Ch**) due to proteolytic activity during incubation with medium. LV-ChABC all generate a diffuse chondroitinase band (GlyCh: partially glycosylated): this migrates more slowly than bacterial chondroitinase, confirming that it has some glycosylation at sites which were not mutated. Some lanes are overexposed as the experiment was principally intended to detect digestion of CSPGs; also see (c). Black bar or star indicates intense bands which partially bleached during imaging. (b) LV-GFP (control) and LV-P. (Top) Probed for NG2; the high-Mr smear due to GAG chains (marked NG2:CSPG), whose distribution varies according to cell type, is all reduced to core protein following LV-P transduction. (Bottom) Probed for chondroitinase ABC. (c) Confirmation of chondroitinase secretion. (1) Commercial chondroitinase (Sigma); (2) medium from HEK cells transduced with LV-C; (3) the same after treatment with N-glycosidase to remove residual glycosylation.