Fig. 2

Effect of patient-derived MLC1 mutants on cellular morphology. a Heterologously expressed GFP, MLC1-GFP, and patient-derived mutants (P92S- and S280 L-GFP) in COS-7 cells were stained with anti-GFP antibody (green, MLC1), GRP78 (blue, ER), and phalloidin (magenta, fibrous actin). filled and empty arrowheads indicate branched actin networks and fibrous actin bundles, respectively. Scale bar: 25 μm (upper) and 5 μm (bottom). The number of filopodia per cell (b) and average length of filopodia (c) in cells transfected with GFP (n = 27 cells and 763 filopodia), MLC1-GFP (n = 24 cells and 1788 filopodia), P92S-GFP (n = 13 cells and 425 filopodia), and S280 L-GFP (n = 17 cells and 281 filopodia) were analyzed by FlioQuant. d Surface expression of wildtype and mutant MLC1 in COS-7 cells was analyzed via surface biotinylation (total = 10% of surface). Tubulin was used as a loading control and a cytosol-specific marker. Transferrin receptor (TfR) was used as a surface-specific marker. e Densitometric analysis of surface MLC1 expression for wildtype and mutant constructs. Levels of fractional surface expression (surface expression/total protein expression) of tested MLC1 constructs (n = 3) were normalized to that of wildtype value (n = 3)