Nucleolin is required for intracellular phosphorylation of FGF1.

A) U2OSR1 cells were transfected with siRNA as indicated, serum starved for 24 h and labelled by [³³P]phosphate, and thereafter stimulated with 100 ng/ml unlabelled, recombinant FGF1 in the presence of 10 U/ml heparin, and 10 nM BafA1 where indicated, for 6 h. FGF1 was extracted from total cell lysates by adsorption to Heparin-Sepharose and analyzed by SDS PAGE and fluorography to detect in vivo phosphorylation, and immunoblotting (IB) to detect FGF1. The lysates were also analyzed for nucleolin and HSP90 by IB. B) U2OSR1 cells transfected with siRNA as indicated were serum starved for 24 h, stimulated with FGF1 and heparin for the indicated time and then lysed and fractionated into a cytoplasmic (CP) and a nuclear (N) fraction before analysis by SDS-PAGE and IB for phospho-PKCδ and total PKCδ. The fractions were also analyzed for nucleolin, lamin A, and ERK1/2 by IB. C) Recombinant FGF1 or FGF1 mutants as indicated, were incubated with recombinant, active PKCδ and [γ-³³P]ATP and thereafter analyzed for in vitro phosphorylation by fluorography, and IB to show loading. D) U2OSR1 cells were serum starved and labelled with [³³P]phosphate and stimulated with FGF1 or FGF1 mutants as indicated for 6 h. The cells were fractionated into cytoplasmic and nuclear fractions. FGFs were extracted from the fractions by binding to Heparin-Sepharose and analyzed by fluorography to detect in vivo phosphorylation and immunoblotting (IB) to detect FGFs. Fractions were also analyzed for marker proteins by IB as indicated. E) U2OSR1 cells were transfected with siRNA as indicated, serum starved for 24 h and labelled with [³³P]phosphate and stimulated with FGF1 in the absence or presence of 1 µg/ml thapsigargin. The cells were fractionated and analyzed as described in (D).