Western blot analysis of EGFR and p-EGFR expression in transfected HCEC.

Total extracted protein from HCEC transfected with pre-miRNA precursors or their inhibitors (antagomirs, AM) and Cy3-labeled pre-miRNA (control) was separated on gradient SDS-PAGE gels, transferred to nitrocellulose and probed with antibodies to EGFR or p-EGFR. Antibodies to β-tubulin or β-actin were used as equal loading controls and for semi-quantitation. In both wounded and non-wounded HCEC, miR-146a mimic treatment decreased whereas its antagomir increased protein levels of p-EGFR (A,B). MiR-424 mimic and antagomir treatments did not change the expression of p-EGFR in non-wounded cells (A,B). However, in wounded cells, miR-424 mimic decreased whereas its antagomir increased protein levels of p-EGFR (A,B). In both wounded and non-wounded HCEC, miR-146a mimic treatment decreased whereas its antagomir significantly increased protein levels of total EGFR (C,D). MiR-424 mimic and antagomir treatments did not significantly change the expression of EGFR both in wounded and non-wounded cells (C,D). Band intensities were quantified using ImageJ software and plotted relative to the loading controls. Blots in A were developed with alkaline phosphatase system, blots in C, with ECL reagent. *, p < 0.05.