Both CD59 clusters and GM1 clusters induced by the sequential additions of CTXB and its polyclonal Abs (Ab-CTXB-GM1 clusters) induced Erk phosphorylation (activation). Note that the simple clustering of five GM1 molecules by CTXB (CTXB-5-GM1) failed to trigger ERK activation. Western blotting was performed by using antiphosphorylated Erk Abs (top) with anti-H-Ras Abs as the loading controls (bottom). The addition of 20 nM EGF was used as a positive control for Erk activation.