JE inhibits SDF-1–induced migration of 70Z/3 cells because of inhibition of ERK phosphorylation. (A) Transwell migration. 70Z/3 cells were pretreated with or without JE for 30 minutes. The cells were then washed, and transwell assay was preformed as described in Figure 2. The results presented are representative of 3 different experiments. (B) Actin polymerization. 70Z/3 cells were pretreated with or without JE for 30 minutes. The change in the cells' actin polymerization was then evaluated as described in panel A. The results presented are representative of 3 different experiments. Error bars represent the standard error of the results of all the experiments used to calculate the average. *Significant at a 98% level. (C) 70Z/3 cells pretreated in the presence (dotted line) or absence (dark line) of CCL2/JE (1 ng/mL). The cells were labeled, and calcium mobilization was monitored before and after SDF-1 stimulation. The results are representative of 3 different experiments (D) 70Z/3 cells were pretreated with or without CCL2/JE (1 ng/mL) for 30 minutes. The cells were then stimulated with SDF-1 for 1 minute. Cells were lysed immediately, and lysates were separated on reducing 10% (wt/vol) SDS-PAGE and immunoblotted with anti-phosphospecific ERK1/2 (p-ERK). Immunoblots were stripped and reprobed with anti-ERK1/2. Ratio calculation is as follows: The intensity of p-ERK band in each treatment was divided by the intensity of the ERK band in each lane. The ratio in the absence of any treatment was normalized to 1, and the ratio in each treatment was calculated as the intensity in treatment relative to 1.