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Figure S1. Phospho- S273-paxillin antibody is specific and phospho- S273-paxillin levels are up-regulated during cell spreading. (a) Phospho- S273-paxillin antibody is specific. Lysates from CHO-K1 cells expressing S273A- or S273D-paxillin-GFP were immunoblotted using anti- phospho- S273-paxillin antibody (top) and anti-paxillin antibody (bottom). The phospho- S273-paxillin antibody specifically detects S273D-paxillin (left) around molecular mass of ∼98 kD, whereas S273A-paxillin (right) was not detected. (b) Phospho-peptide competition. Immunoblots were performed on CalA- treated CHO-K1 lysates using phospho-S273-paxillin antibody dilutions preincubated with 200-fold molar excess of no peptide (left), phospho- S273-paxillin (middle), or non phospho- S273-paxillin peptide (right). No signal was detected in the presence of the competitive phosphopeptide (middle), whereas the signal was retained in the presence of the nonphosphopeptide (right), confirming the antibody specificity. (c) Time course of S273-paxillin phosphorylation during cell spreading. Immunoblots were performed on CalA-treated CHO-K1 lysates from cells in suspension and those allowed to spread under migration-promoting conditions for different time intervals (0-3.5 h) using anti-phospho-S273-paxillin (top) and anti-paxillin antibody (bottom). Low levels of phospho-S273-paxillin were detected in suspended cells with an increase after 1 h of plating that was sustained until 3.5 h after plating, indicating that the S273-paxillin phosphorylation is up-regulated during cell spreading. (Cell spreading initiated ∼1 h after plating.) (d) Densitometric quantification of normalized phospho-S273-paxillin levels during cell spreading, reached the maximum range at 1-2.5 h after plating. Error bars represent SEM.