FIGURE 3.

Paxillin Ser-85 phosphorylation is important for haptotactic cell migration. A and B, cells were transfected with mock or HA₃-paxillin (WT or S85A) constructs for 36 h before a Transwell migration assay, in which the lower chamber was filled with 1% BSA and DMEM-H (negative control), 10% FBS, or serum-free medium containing 10 μg/ml collagen I (Coll) for 12 h. After incubation, migrated cells on the chamber filter were stained with Diff-Quik solution (Medion Diagnostics), and at least five random images for each condition (A) were imaged to calculate means ± S.D. (B). C, cells were transfected with HA₃-paxillin (WT or S85A) for 36 h and kept in suspension (Sus) or replated onto collagen I-precoated culture dishes for 30 min in the presence of 10% FBS before harvesting and analysis of whole cell lysates by immunoblotting for the indicated molecules. D and E, assay for cell migration through the Transwell for 6 h was performed as described for A. Cells were pretreated with SB202190 (SB) 30 min before loading cells into the upper chambers. Migrated cells from representative images (D) were calculated for means ± S.D. (E). F, cells were transfected and manipulated as described for C in the absence (−) or presence of SB202190 pretreatment before harvesting and analysis of whole cell lysates by immunoblotting for the indicated molecules. The data shown represent three independent experiments. pS⁸⁵Pax, phospho-Ser-85 paxillin.