Figure 4

Expression and subcellular localization of wild-type and phosphorylation mutant forms of FGF1. (a) PC12 cells were transfected with the pLK-FGF1^WT, pLK-FGF1^S130A or pLK-FGF1^S130D dexamethasone-inducible vectors to respectively overexpress FGF1^WT, FGF1^S130A or FGF1^S130D. The pLK-FGF1^S130A and pLK-FGF1^S130D vectors were generated by site-directed mutagenesis. (b) Neo, FGF1^WT, FGF1^S130A and FGF1^S130D PC12 cell lines were cultured in the absence or presence of 5 × 10⁻⁷ M dexamethasone for 48 h. FGF1 expression was analyzed by western blot using actin level as a control. The presence of dexamethasone increased FGF1 levels at comparable levels in the different PC12 cells transfected to express one of the different FGF1 forms. (c) After heparin sepharose concentration, FGF1 levels in cell extracts and conditioned media of native, Neo, FGF1^WT, FGF1^S130A and FGF1^S130D PC12 cells were examined. (d) FGF1^WT, FGF1^S130A and FGF1^S130D PC12 cell lines were treated with dexamethasone for 48 h. Nuclear (N) and cytosolic (C) proteins were analyzed by western blot for FGF1, Enolase (cytosolic marker) and Lamin A/C (nuclear marker). Total protein extracts (TE) were used as controls. FGF1 was detected in all the fractions. (e) Quantification of the levels of FGF1 normalized to Lamin A/C levels in the total lysates and nuclear fractions of three independent experiments