FIGURE 4.

Cell binding and Neurocan interaction with NrCAM. (A) COS-7 cells transfected with vector alone (pCAGGS-IRES-mEGFP) or pCAGGS-NrCAM-IRES-mEGFP were pre-treated with 8 nM Neurocan, then fixed and subjected to immunofluorescence staining without permeabilization to detect surface-bound Neurocan (red). Scale bar = 100 μm. (B) Mean fluorescence intensity (±SEM) of Neurocan immunofluorescence staining on the surface of COS-7 cells, as shown in panel A. NrCAM-expressing cells treated with Neurocan showed significantly greater levels of bound Neurocan than untreated cells. Fluorescence intensity in cells with vector alone treated with Fc or Sema3F-Fc was non-significant (ns). ^∗p > 0.05, t-test, n = 5 images each condition. (C) Lysates (50 μg) of cells transfected with vector alone or pCAGGS-NrCAM-IRES-mEGFP were treated with Neurocan as in panel A, and immunoblotted (IB) with Neurocan antibodies. Blots were reprobed with antibodies directed against GAPDH (loading control) or NrCAM (expression control). Representative immunoblots of three experiments are shown. (D) Mouse cortical neuron cultures from NrCAM null mice were transfected with vector alone or pCAGGS-NrCAM-IRES-EGFP, and pre-treated with 20 nM Neurocan before fixation and immunostaining to detect surface-bound Neurocan. In merged images of EGFP (green) and Neurocan (red), more Neurocan immunofluorescence was observed on the surface of neurons expressing NrCAM than on NrCAM null neurons with vector alone. Scale bar = 50 μm. (E) Mean fluorescence intensity (±SEM) of surface-bound Neurocan immunostaining on neurons in panel D NrCAM-expressing cells treated with Neurocan showed significantly greater levels of bound Neurocan than NrCAM-minus neurons. ^∗p > 0.05, t-test, and n = 10 neurons per condition. (F) ELISA of Neurocan-AP or control AP protein binding to NrCAM-Fc or positive control NCAM-Fc on protein A-coated microtiter wells. AP binding was detected colorimetrically with p-nitrophenylphosphate. The mean (±SEM) optical densities (OD 405) of Neurocan-AP bound to NrCAM-Fc or NCAM-Fc were significantly greater than control AP (t-test and ^∗p > 0.05). (G) Recombinant human Neurocan was incubated in Tris buffered saline with purified Fc, Sema3F-Fc, or Sema3A-Fc proteins, then complexes were pulled down with Protein A/G Sepharose beads. Immunoblotting for Neurocan showed no binding of Neurocan to Fc or Sema3F-Fc, whereas Neurocan bound effectively to Sema3A-Fc. Blots were reprobed with anti-Fc antibodies to demonstrate that equivalent amounts of Fc fusion proteins were pulled down. Recombinant Neurocan (left lane) ran as a broad band between 250 and 130 kDa.