Figure 2. CSPGs bind LAR protein with high affinity in COS-7 cells.

A, Schematic diagram indicates the LAR transmembrane protein. LAR ectodomain includes several Ig-like and fibronectin type-III domains. Its intracellular portion consists of two cytosolic phosphatase domains called D1 and D2. B, COS-7 cells were transfected with control, LAR-WT, LAR mutant with D2 domain deletion (DeltaD2) or LAR mutant with D1 domain mutation (C1522S). Forty-eight hrs after transfection, cells were incubated with purified CSPGs and CSPG-binding signal was detected via immune-based staining. Scale: 100 μm. C, Image at high magnification indicates CSPG binding signal to COS-7 transfected with LAR-WT. Scale: 25 μm. D, Immunostaining for LAR indicates LAR expression in COS-7 cells 48 hrs after transfection. Scale: 50 μm. E, Immunostaining for LAR deltaD2 indicates expression of this mutated LAR in COS-7 cells 48 hrs after transfection. Scale: 50 μm. F, Bar graph indicates CSPG binding signal for COS-7 cells in 96-well plates. The binding signal was quantified in each group with a plate reader at 405 nm. n = 6 wells in each group. G, A dose-response curve for CSPG binding was determined in COS-7 cells transfected with LAR-WT. H and I, Bar graphs indicate CSPG binding signals to COS-7 cells quantified from 96-well plates with a plate reader at 405 nm. n = 6–10 wells in each group. CS: CSPG. LAR Ig1M: LAR mutation in the first Ig-like domain. J, The affinity of CSPG interaction with LAR was determined from the supernatants of COS-7 cells via an enzyme-linked immunosorbent assay. K, Western blot indicates expression of LAR protein in COS-7 cells transfected with LAR-WT, but not in the control. COS-7 cells transfected with deltaD2 mutation exhibit a reduced band size for LAR. L, LAR conjugated to c-myc in COS-7 supernatants was coimmunoprecipitated with CSPG neurocan using antibodies against c-myc (IP) and neurocan (blot). In F–J, *p<0.05, **p<0.01, Student’s t-test.