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Fig. 2. ERK5 is localized in the nucleus before and after stimulation. HeLa cells (A) or Rat-1 cells (B) were grown on coverslips, serum-starved and activated with 50 ng/ml EGF for the indicated times. The treated cells were then fixed and stained with anti-ERK5 antibody (N-19). Then the cells were tested for ERK5 phosphorylation. For this purpose, HeLa cells (C) or Rat-1 cells (D) were grown in 6 cm tissue culture plates and activated with 10 ng/ml EGF for the indicated times. After treatment, the cells were harvested with RIPA buffer, and cell lysates were separated by SDS-PAGE and subjected to western blots analysis with anti-ERK5 or anti-phospho-ERK5 (P-ERK5) antibodies. Biochemical fractionation of ERK5 was performed as described under Materials and Methods. (E) HeLa cells (upper panels) or Rat-1 cells (lower panels), were grown in 10 cm tissue culture plates, serum-starved and activated with EGF (50 ng/ml, 15 minutes). Then the cells were washed with ice cold PBS and fractionated into nuclear and cytoplasmic fractions. Equal volume aliquots of each fraction were subjected to SDS-PAGE and western blotting and analyzed with anti-ERK5 (C terminus) and anti-phospho-ERK5 (P-ERK5) antibodies. (F) The samples described in E were subjected to western blot analysis with anti-Sp-1 and anti-caspase 3 antibodies.
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