Fig. 3

RT-PCR analysis of HAMP gene, and RT-PCR and western blot analysis of Fpn1 expression levels. HepG2 cells were treated for 16 h in serum-free medium with 300 μM BCS and/or 40 ng/ml IL-6. a after 16 h of treatment, RNA was isolated, reverse transcribed and subjected to PCR. The amplicons relative to HAMP and β-actin genes were analysed by agarose gel electrophoresis and intensity of bands was determined by ImageJ 1.47v software (http://imagej.nih.gov/ij). The values of intensity relative to HAMP were normalized by using the β-actin housekeeping gene. b densitometric analysis of HAMP gene RT-PCR results (c) representative image of Fpn1A isoform RT-PCR product analysed as described above and (d) densitometric analysis relative to Fpn1A RT-PCR results. The values were normalized by using the β-actin housekeeping gene. e representative image of Fpn1B isoform RT-PCR product analysed as described above and (f) densitometric analysis relative to Fpn1B RT-PCR results. The values were normalized by using the β-actin housekeeping gene. g representative image of Fpn1 protein immunoblot result relative to membrane proteins extracts. Equal amounts of proteins were loaded per lane. h densitometric analysis of Fpn1 protein. The values are normalized to β-actin protein level. All values are expressed as means ± SEM (n = 6). All indicated differences were statistically significant (p < 0.05). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Expression levels of control condition were normalized to one, and all values are expressed as relative units