Secondary antibody labeled with peroxidase

Cell supernatants were collected from 24-well plates and BCA assays (ThermoFisher Scientific, Madrid, Spain) were performed to calculate protein concentration of samples. Protein concentration variability between samples was low (4.8–5.5 μg/μL for EAhy.926 and 1.3-1.6 μg/μL for HUVEC). Normalization in the western blot was based on the amount of protein loaded. For this purpose, 100 μg (EAhy.926) and 30 μg (HUVEC) of total protein was separated by SDS-PAGE in reducing conditions by electrophoresis in 8% polyacrylamide gels. Gels were transferred onto PVDF membranes (Amersham Hybond P 0.45, GE Healthcare, Barcelona Spain) and blocked 1 hour in 5% w/v non-fat drymilk. Antibody against human TPFI (#ADG72, Sekisui Diagnostics, Rüsselsheim, Germany) was used 1:5,000 dilution and incubated overnight at 4 °C. The secondary antibody labeled with peroxidase (GE Healthcare, Barcelona, Spain) was used 1:10,000. ECL Prime Detection Kit and ImageQuant LAS 4000 Imager (GE Healthcare, Barcelona, Spain) were used for sample detection. Densitometric analyses were performed with ImageJ software (http://rsb.info.nih.gov/ij/). Linearity between the amount of sample and densitometry was assessed (r² = 0.95). Data were expressed as changes relative to cells transfected with SCR, taken as 100%.

Transfected HepG2 cells were washed twice with phosphate-buffered saline (PBS). They were then lysed in RIPA buffer [150 mMNaCl, 1 mMethylenediaminetetraacetic acid, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 20 mM 3-(N-morpholino) propanesulfonic acid (MOPS), 1 mMphenylmethylsulfonyl fluoride, pH 7.0] on ice for 20 min. The lysates were centrifuged at 12,000×g for 5 min at 4°C and the supernatants were collected for analysis of the protein concentration using a Bicinchoninic Acid (BCA) Protein Assay (Sigma-Aldrich, Madrid, Spain). These lysates (each 20 μg) were blotted and immunostained with different monoclonal antibodies: anti-HNF4α (ab92378; Abcam, Madrid, Spain) and anti-GAPDH (ab128915; Abcam, Madrid, Spain). HNF4α and GAPDH were immunodetected with the appropriate secondary antibody labeled with peroxidase (GE Healthcare, Barcelona, Spain). Western blotting detection, their corresponding densitometric analysis, and the expression of data were performed in a manner similar to that previously described [33 (link)].