Heparin inhibits HGF-induced p-Met, p-MAPK, MMP-2 and MMP-9 activation in SK-HEP-1.

In order to examine the time dependent effects of HGF, overnight starved SK-HEP-1 cells were grown in presence or absence of HGF for the time periods indicated. Total cell lysates were then analyzed by immunoblotting. Blots were probed with anti-p-Met, anti-c-Met, anti-pERK1/2, anti-ERK1/2 and anti-calnexin antibodies. From top to bottom: HGF stimulated c-Met phosphorylation; the amounts of c-Met protein present whole cell lysates; HGF induced ERK1/2 phosphorylation and ERK1/2 and c-Met total protein levels. Membranes were re-probed with ERK1/2 and c-Met antibody after stripping. The bottom panel verifies equal protein loading among the lanes (3A). SK-HEP1 cells left untreated or treated with HGF for 2 hours in the presence and absence of heparin. Total protein lysates were analyzed by immunoblotting. Membranes were blotted with anti-p-Met, anti-c-Met, anti-pERK1/2, anti-ERK1/2 antibodies, and anti-calnexin antibodies. The first panel shows the level of tyrosine phosphorylated c-Met; the amount of c-Met protein present in the each lane is shown in the second panel. The third and fourth panels show the amount of phosphorylated ERK1/2 level and the level of ERK1/2 protein in the whole cell lysates, respectively. Membranes were re-probed with ERK1/2 and c-Met antibody after stripping. The lower panels show the amount of calnexin as a loading control (3B). Zymographic gel showing active MMP-9 and MMP-2 bands in 24 h conditioned medium from cultured SK-HEP-1 cells left untreated or stimulated with HGF and heparin (3C, 3D).