Expression of eGFP WASp in WASp-null cells results in the reconstitution of the normal organization of the actin cytoskeleton. Bone marrow-derived osteoclasts from normal (SV129), WASp-null (WASp-/-), or WASp-null transduced with eGFP (WASp-/-eGFP) or eGFP-WASp (WASp -/-eGFP-WASp) were lysed in RIPA buffer at day 7 after plating and subjected to a 10% SDS-PAGE gel electrophoresis, probed with anti-WASp (A) or anti-eGFP antibodies (B). Signal was detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection system. Visualization of eGFP required a 10-second film exposure, whereas visualization of eGFP-WASp required 90 seconds of exposure. Blots were stripped with 2% SDS and 0.7% β-mercaptoethanol for 1 hour at 50°C and reprobed for β-actin to check the total amount of protein loaded per lane. WASp-null bone marrow osteoclasts derived on glass coverslips (C) or bone slices (D) and transduced with eGFP (▦) or eGFP WASp (▪) were fixed with 3% paraformaldehyde, permeabilized with 0.05% Triton X-100, and the presence of actin filaments and vinculin was detected by immunofluorescence. We quantified the percentage of cells displaying each of the identified arrangements of actin filaments in 100 cells per experiment. The graphs illustrate the mean percentage of cells ± SD from 3 experiments.