Agrin delivers signals through the α-DG. (A) Control and Musk-L;Agrn^−/− splenocytes were incubated with (10 μg/mL) or without (0 μg/mL) the anti-α-DG blocking antibody IIH6C4 or the same concentration of its isotype control (IgM). Annexin⁺/PI⁺ analysis was performed on CD11b⁺/F4/80⁺ macrophages after 24 hours (ctrl, n = 10; Musk-L;Agrn^−/−, n = 6.) Bars with different letters are significantly different from each other (student-Newman-Keuls test, P < .05). (B-C) Representative confocal images (scale bar, 5 μm) of macrophages isolated from P5 control mice and quantitative colocalization analysis. (B) Cells were fixed and stained for α-DG (green), Grb2 (red), and DNA (blue). Corresponding pictures were merged (yellow). Each colocalized pixel was plotted on a scatter diagram to give correlation plots, with colocalizing pixels falling around the diagonal line. Data from at least 30 cells were used to calculate the Pearson coefficient (P) as mean ± SEM. (C) Cells were incubated with primary mAbs to α-DG, CD71, or MHCII and then secondary mAbs (green) to induce patching. Fixed cells were stained for Grb-2 (red) and DNA (blue). Corresponding pictures were merged (yellow). Percentage of colocalized volume and the Mander colocalization coefficient with Grb2 were determined as described in “Immunochemistry and immunofluorescence.” Data from at least 30 cells were used. (D) Total splenocytes were stimulated with commercial agrin (1153-1959; black line), or laboratory-produced agrin (1379-1940; blue line) and the phosphorylation of Erk 1/2 was evaluated in CD11b⁺ /F4/80⁺ cells. (E) Total splenocytes preincubated with the anti–α-DG blocking antibody IIH6C4 or its isotypic control (IgM) were stimulated with commercial agrin (1153-1959) for 15 minutes and the phosphorylation of Erk 1/2 was evaluated in CD11b⁺ /F4/80⁺ cells. Changes in Erk phosphorylation were expressed as fold increase of MFI of stimulated over unstimulated cells (time 0); n = 3. Bars with different letters are significantly different from each other (Student Newman-Keuls test, P < .05).