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Fig. 3. Inhibition of FGF-1 translocation by bafilomycin A1 and FGF-1 accumulation in the nucleus by LMB treatment. (A) COS-1 cells were transfected with FGFR1 or FGFR4, treated with [³³P]phosphate and incubated for 6 hours with FGF-1 and heparin in the presence or absence of bafilomycin A1 and monensin, as indicated. FGF-1 was extracted from the cell lysates and assessed for phosphorylation by SDS-PAGE and autoradiography. (B) COS-1 cells were transfected with FGFR as indicated, pretreated with [³³P]phosphate and incubated for 6 hours with FGF-1 or FGF-1 K132E and with LMB where indicated. The cells were fractionated into cytoplasmic (C) and nuclear (N) fractions and FGF-1 was extracted as described in Materials and Methods and then separated by SDS-PAGE and blotted onto Immobilon-P membrane. ³³P-phosphorylated FGF-1 in the cytoplasmic (upper panel) and nuclear (second panel) fractions was detected by autoradiography and the total cell associated FGF-1 in the cytoplasmic (third panel) and nuclear (bottom panel) fractions was detected by anti-FGF-1 antibodies. (C) Cytoplasmic and nuclear fractions of lysed COS-1 cells were analyzed for their purity by western blotting and antibodies against the cytosolic protein MAPK, the ER resident protein Calnexin and the nuclear protein LaminA. (D) The experiment was performed as described in (B) but only nuclear fractions were analyzed. In addition, known amounts of FGF-1 was loaded on additional lanes of the gel to estimate the amount of FGF-1 that had been extracted from the nuclear fraction. Upper panel, autoradiography. Lower panel, anti FGF-1 immunodetection.

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