Figure 3

Comparison of brevican, the ADAMTS-derived neoepitope of brevican and WFA reactivity in rat and mouse brain extracts. Western blot of brevican, EAV(M)ESE, and Wisteria floribunda agglutinin (WFA) in rodent brain extracts before and after chondroitinase digestion: Rat (Rt) and mouse (Ms) extracts were probed for (A) anti-brevican, (B) anti-EAVESE (Rt) or anti-EAMESE (Ms), or (C) biotinylated WFA: Samples were treated with (+) and without (-) Chondroitinase ABC (Chase). (A+) The 145 kD core protein of brevican increased after Chase treatment, (B+) the proteolytic brevican fragment remained unchanged, and (C+) only a high molecular weight WFA-reactive band was diminished in Ms. (C) After probing with WFA, multiple, unidentified lower molecular weight bands were observed along with less abundant, high molecular weight moieties. The right panel in (C) was probed with secondary, HRP-conjugated streptavidin alone, which revealed two, major non-specific bands. (D) After differential centrifugation of rat brain tissue, brevican immunoreactivity (left panel) was predominately found in the soluble fraction (S), whereas most of the WFA reactivity (right panel) was observed in the membrane "insoluble" fraction (I) whereas anti-EAVESE immunoreactivity (middle panel) was evident in both fractions. (E) Rt and Ms samples were treated with Chase in the absence (-) and presence (+) of a protease inhibitor cocktail (left panel) and probed with biotinylated-WFA. The high molecular weight smear is eliminated after treatment with Chase, but the protease inhibitor did not change the pattern. (right panel) The same membrane was probed with anti-brevican where complete removal of CS chains led to an increase in abundance of the core protein with no change in the abundance of fragment. Protease inhibitor had no effect on Chase action.