Fig. 7. Nuclear translocation of GST-ERK in vitro is affected by calcium. (A) HeLa and Rat1 cells were serum-starved (16 h 0.1% FBS), washed in ice-cold buffer as described [45] and permeabilized with digitonin (60 µg/ml, Calbiochem) for 5 min on ice. Cells were then washed and incubated with 0.3 mg/ml of recombinant GST-actERK2 (eluted from the glutathione beads with reduced glutathione) for 30 min at room temperature. The cells were then fixed with 3% PFA at 23°C, and methanol/acetone (50% vol/vol 30 min, 23°C). After fixation, the cells were stained with anti-gERK Ab as above. (B) Recombinant NUP153 (0.5 µg/binding assay) was incubated (2 h, 4°C) with GST-ERK2 conjugated to glutathione beads in the absence or presence of increasing calcium concentrations as indicated. After incubation, the beads were washed three with buffer A+0.15M NaCl containing the indicated calcium concentration and this was followed by boiling in sample buffer and Western blot with anti-gERK and anti-NUP153 Abs.