FIGURE 6. Cat-315 and 6B4 reactive perineuronal nets are present in the cortex of adult POMGnT1 knockout animals.

Coronal sections from the barrel cortex of adult POMGnT1 +/+ and −/− samples were immunostained and counter-stained for Hoechst nuclear stain (blue). A, POMGnT1 +/+ section stained for Cat-315 (red) shows reactivity that is localized to PNs in layer IV and VI. B, POMGnT1 −/− sections stained for Cat-315 shows abnormal cortical lamination but the presence of Cat-315 reactive PNs. C, POMGnT1 +/+ section stained for 6B4 (red) shows reactivity that is localized to PNs. D, POMGnT1 −/− section shows abnormal cortical lamination but the presence of 6B4 (red) reactive PNs. Bar, 100μM. E, chABC treated soluble adult brain homogenates from POMGnT1 +/+, +/− and −/− mice were analyzed by Western blot analysis for Cat-315, HNK-1, 5210, aggrecan, 3F8, H300 and brevican reactivity. Reactivity and molecular weight for Cat-315, HNK-1, 5210 and aggrecan reactive bands appeared unaffected in the POMGnT1 −/− tissue relative to controls (+/+ and +/−). In contrast, 3F8 reactivity was absent in POMGnT1 −/− samples and the apparent molecular weight of the H300 bands were shifted downwards. Reactive bands are isolated from the same region of the gel, as reactivity for Cat-315, HNK-1, 5210, aggrecan, 3F8 and H300 were all present above 250kD. As in previous figures, brevican reactive bands were located at 150kD. F, adult brain soluble fractions from +/− and −/− animals were subject to immunoprecipitation (IP) with the Cat-315 antibody. Immunoprecipitated material was then detected with antibodies against either aggrecan or H300. Aggrecan reactive bands were pulled down with the Cat-315 antibody and this reactivity was essentially unaltered in −/− samples compared to +/− controls. In contrast, a band reactive for H300 was only detected in +/− samples and absent from −/− samples. (+) denotes the inclusion of the Cat-315 antibody in IP reaction, (M) indicates mock control IP reactions lacking Cat-315 antibody.