Fig. 5.

The Cat-315 CSPG is a constituent of the extracellular matrix. Cell homogenates were separated into particulate and soluble fractions by centrifugation. The particulate fraction was rinsed several times by resuspension in PBS followed by centrifugation. The washed particulate fraction was subjected to extraction with various reagents and then separated again into supernatant and particulate fractions. These final supernatant and particulate fractions were Western blotted and probed with Cat-315; eachlane contains 25 μg of protein. Neither PBS (lanes 1, 2) nor Triton X-100 (lanes 3, 4) released Cat-315 immunoreactivity from the particulate into the soluble fraction. Na₂CO₃ (lanes 5,6) partially released Cat-315 immunoreactivity from the particulate to the soluble fraction. Together with the ability of Cat-315 to stain the surfaces of unfixed, unpermeabilized cells, these data indicate that at least a portion of the Cat-315 CSPG is entirely extracellular and peripherally attached to the neuronal surface and thus is a constituent of the extracellular matrix.P, Particulate fraction; S, soluble fraction.