Figure 4.

The PDZ-binding motif of SALM1 plays an important role in SALM1 surface targeting and the recruitment of PSD-95. A, The deletion of the PDZ-binding motif of SALM1 changes the level of the surface expression of SALM1 in HeLa cells and in neurons. HeLa cells (a, b) and neurons (d, e) were transfected with SALM1-myc (a, d) or SALM1-Δ4myc (b, e). Neurons were also transfected with GFP (d, e; green fluorescence). Surface staining with anti-SALM1 N-terminus antibody is shown in red (left panels), and permeabilized staining with anti-myc is in green (a, b) or blue (d, e) for total SALM1 (middle panels). Surface expression and total expression were analyzed using MetaMorph software and graphed by Excel (Microsoft, Seattle, WA) (c, f). The ratio of surface to total staining for full-length SALM1 was arbitrarily assigned 100%. The surface staining of cells transfected with SALM1-Δ4myc is dramatically reduced when compared with that of cells transfected with SALM1-myc (11.0% in HeLa cells and 19.8% in neurons). Neurons transfected with SALM1-myc show larger puncta than those transfected with SALM1-Δ4myc. The size of puncta is 58.1 ± 14.0 pixels with transfection of SALM1-myc and 29.5 ± 9.6 pixels with transfection of SALM1-Δ4myc. **p < 0.001. Scale bar, 10 μm. Data were obtained from 14 DIV cultures 48 h after transfection. For SALM1-myc and SALM1-Δ4myc, results presented in f are based on n = 22 and 5, and in g on n = 17 and 12, respectively; values are mean ± SD. B, SALM1 is colocalized with PSD-95. Neurons at 14 DIV were transfected with SALM1-myc or SALM1-Δ4myc, and, 48 h after transfection, cells were fixed and stained for endogenous PSD-95. The percentage of area colocalization for SALM1-myc and PSD-95 is 57.8 ± 3.7% and for SALM1-Δ4myc and PSD-95 is 16.1 ± 4.9%. The area of colocalization is defined as the percentage of SALM that has PSD-95 staining. **p < 0.001. Scale bar, 10 μm. For SALM1-myc and SALM1-Δ4myc, results presented in c are based on n = 14 and 24, respectively; values are mean ± SD.