Fig. 3. ROK and Bcl-2 are unable to suppress vacuoles accumulation in cells expressing Gas3/PMP22. (A) The indicated ROK constructs were co-expressed with gas3/PMP22-GFP in NIH3T3 cells. After 24 hours from microinjection cells were fixed and processed for immunofluorescence to detect Gas3/PMP22-GFP and ROK as described in Materials and Methods. Cells were scored for reduced spreading as previously described (Brancolini et al., 1999). Data represent arithmetic means±s.d. for four independent experiments. (B) Immunofluorescence analysis of NIH3T3 cells co-expressing gas3/PMP22-GFP and ROK1–1271 or Gas3/PMP22-GFP and ROK K112A. NIH3T3 cells 24 hours after seeding were microinjected with pEGFP-N1-gas3/PMP22 (20 ng/µl) and pXJ40- ROK1–1271 (80 ng/µl) or with pXJ40- ROK K112A (80 ng/µl). After 24 hours cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22 and HA-tagged ROK. Bar, 18 µm. (C) Gas3/PMP22-GFP and hPLAP, as controls, were overexpressed in REF 52 cells. After 21 hours from microinjection 10 µM of Y-27632 (final concentration) was added to the culture medium and cells were fixed 1 hour later and processed for immunofluorescence to score the Gas3/PMP22 phenotype. Data represent arithmetic means±s.d. for four independent experiments. (D) Immunofluorescence analysis of REF 52 cells expressing Gas3/PMP22-GFP or hPLAP treated or not with Y-27632. REF 52 cells 24 hours after seeding were microinjected with pEGFP-N1-gas3/PMP22 (20 ng/µl) or with pGDSV7S-hPLAP (80 ng/µl). Bar, 18 µm. (E) Gas3/PMP22-GFP and the indicated genes were co-expressed in NIH3T3 cells. After 18 hours from microinjection cells were fixed and processed for immunofluorescence to visualize Gas3/PMP22-GFP and the co-expressed protein. Cells were scored for accumulation of vacuoles. Data represent arithmetic means±s.d. for four independent experiments.