Increased phosphorylation of ERK1/2 in DAOY and D556 cells in response to exogenous β2m. A) Serum starved DAOY cells were incubated in the presence or absence of human β2m (2 μg/ml). At indicated times after β2m addition, cells were lysed and the lysates were analyzed by Western Blotting for phosphorylated (pERK) and total ERK, as well as for GAPDH as a control for loading. B) DAOY cells were treated with decreasing concentrations of β2m, and analyzed for phosphorylation of ERK1/2 15 minutes post β2m addition as in (A). Densitometric quantification of pERK1/2 bands relative to GAPDH in each of the treatments is shown below representing 3.65- (2 μg/ml), 2.65- (0.4 μg/ml), and 1.44- (0.08 μg/ml) fold increase in pERK1/2 over the background observed in the absence of β2m. C) β2m (2 μg/ml) was mixed with anti-β2m antibody (0.2 mg/ml) prior to the addition to DAOY cells and ERK1/2 activation was analyzed 15 minutes post β2m addition. Densitometric quantification is shown below indicates 43% inhibition by anti-β2m antibodies of β2m-induced ERK1/2 phosphorylation. D-E) The effect of β2m (2 μg/ml) incubation of indicated time lengths on phosphorylation of ERK1/2 in D556 (D) or D283 (E) cells was examined by Western blotting. F) Summary of pERK1/2 quantification in three different cell lines at indicated times after β2m treatment, relative to the levels in untreated cells (results are expressed as fold induction).