Fig. 2.

a Western blot analysis in whole cell lysates and media of HEK293 cells 48 h after transfection with wild type, R1698C or L1176P agrin. Levels of full length agrin (~250KDa) in the media were compared in the bar chart, n = 3. Alpha tubulin (~50KDa) was used as a loading control for cell lysates and DsRed monomer (~27 kDa) as a marker to verify transfection efficiency; (b) 48 h post HEK293 cell-transfection cells were incubated in cycloheximide (CHX) (20 μg/ml) for the indicated times above the collumns, the mutant agrin showed a time-course degradation not seen for wild type, n = 3; (c) Representative image of myotubes labelled with α-Butx-594 exposed to either wild type, R1698C, L1176P agrin for 16 h in media containing an equivalent agrin concentration. Magnification: 20×. The scale bar represents 10 μm. Bar charts showing the number and average size of AChR clusters per field, n = 3. (Data represents the mean ± SD of three experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)