Fig. 1

FGFR2c aberrant expression and signaling induce PKCε activation. HaCaT clones stably transduced with pBp-FGFR2c or with pBp-FGFR2b or empty pBp vector, used as controls, were left untreated or stimulated with FGF7 or with FGF2 in presence or absence of the FGFR tyrosine kinase inhibitor SU5402 as described in Material and Methods. a Western blot analysis shows that the 140 KDa band corresponding to the molecular weight of FGFR2 is mainly evident in cells transduced with FGFR2b or FGFR2c isoforms. An appreciable phosphorylation of PKCε at the autophosphorylation site Ser 729 is observed only in pBp-FGFR2c clones upon FGF2 stimulation and this effect is abolished by SU5402. A moderate up-regulation of PKCε protein level is detectable only in pBp-FGFR2c clones, particularly in response to FGF2. A moderate phosphorylation of PKCδ at the autophosphorylation site Serine 645, is visible in all clones in response to FGF7 stimulation, while no evident modulation of PKCδ protein level is observed. Equal loading was assessed for p-PKCε with the anti-PKCε antibody, for p-PKCδ with the anti-PKCδ antibody, for PKCε and PKCδ with the anti-actin antibody. For the densitometric analysis, the values from 3 independent experiments were normalized, expressed as fold increase and reported in graph as mean values ± standard deviation (SD). Student t test was performed and significance levels have been defined as p < 0.05: *p < 0.05, ** p < 0.01. b Real time RT-PCR analysis shows a positive modulation of PKCε expression at mRNA transcript level only in pBp-FGFR2c clones, particularly in response to FGF2. No evident modulation of PKCδ mRNA expression is observed. Results are expressed as mean value ± standard error (SE). Student’s t test was performed and significance levels were defined as p < 0.05: *p < 0.05, *** p < 0.001