Figure 4. Determination of protein(s) binding to the 5’-UTR of the OPN promoter region using gel-shift assays.

(A) Cytoplasmic protein lysates were extracted from HepG2 and Hep3B cells and incubated with (α-³² P) ATP (2500 Ci/mmol) end-labeled synthesized 5’-UTR from the human OPN promoter sequence using T4 polynucleotide kinase followed by G-50 column purification. The final products were resolved on 6% poly-acrylamide gel electrophoresis using 0.5× Tris/borate EDTA buffer and visualized by autoradiography. Gel is representative of three experiments.

(B) Full-length OPN 5’-UTR or deletion in OPN 5’-UTR (58–157) along with OPN promoter region, OPN coding region and 3’-UTR were ligated to the pcDNA3.1 expression vector at the C-terminal followed by transient transfection of HepG2 and Hep3B cells and assayed for OPN protein expression by Western blot analysis. Blot is representative of three experiments.