Fig. 6. Calcium regulates ERK nuclear association, but not its activation. (A) EGTA does not affect tyrosine phosphorylation during BCR signaling. WT DT40 cells were either not stimulated (lane 1) or stimulated with anti-IgM for 5 min in the absence (lane 2) or presence (lane 3) of 1 mM EGTA, and protein tyrosine phosphorylation by anti-phosphotyrosine western blotting. (B) ERK activation is not affected by EGTA. WT DT40 cells were either not stimulated (lane 1) or stimulated for the indicated times with anti-IgM in the absence (lanes 2–7) or presence (lanes 9–14) of 1 mM EGTA, and lysates probed for activated ERK (anti-phospho-ERK, top panel), or total ERK (bottom panel) by western blotting. Lane 8 contains lysates from cells treated with EGTA but not stimulated. (C) ERK nuclear association is affected by EGTA. WT DT40 cells were either not stimulated (top left panel) or stimulated for 10 min with anti-IgM in the absence (bottom left panel) or presence (bottom right panel) of 1 mM EGTA and stained for ERK as described in Materials and methods, then analyzed by confocal microscopy. The top right panel contains cells treated with EGTA but left unstimulated. In each panel, the left side is a confocal fluorescent image and the right is a phase contrast image of the same cells.