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. 2010 Nov 29;286(17):15428–15439. doi: 10.1074/jbc.M110.185058

FIGURE 3.

FIGURE 3.

Plasmin induces serine phosphorylation of A2 at positions 11 and 25. A, HEK293 cells were stably transfected with either a Myc-tagged annexin A2 wild type plasmid or Myc-tagged S11A, S25A, or S11A/S25A mutant plasmids. Then total cell lysates were examined either by phosphoserine immunoprecipitation followed by anti-A2 immunoblot analysis or by immunoblotting with anti-phospho-PKC-, anti-PKCα-, anti-phospho-ERK1/2-, or anti-ERK1/2-specific IgGs. Total transfected A2 was estimated by immunoblotting with an anti-Myc IgG, and β-tubulin was employed as a loading control. B, CMECs were stimulated with plasmin (15 min). Total lysates were immunoprecipitated (IP) with anti-p11 IgG and immunoblotted with anti-A2 IgG or anti-p11. Total lysates were also immunoprecipitated with anti-ubiquitin and immunoblotted with anti-p11, anti-phospho-PKC, and anti-phospho-ERK1/2 IgGs. Cell surface A2, p11, and CD47 were analyzed by cell surface biotinylation. C, the levels of cytoplasmic (A2·p11)2 complex and of surface A2 were quantified by densitometry and presented as the percentage of the signal detected in control cells (defined as 100%). Shown are the mean ± S.D. (error bars) for three separate experiments. D, HUVECs grown on glass coverslips were treated with plasmin for the indicated time periods and washed. Permeabilized cells were stained for cytoplasmic p11 (green), A2 (red), and DAPI (blue) (left two panels). Non-permeabilized cells were stained for surface A2 (red), DAPI (blue), and β-tubulin (green) (right two panels). The absence of signal in control IgG-stained cells indicated the specific staining. E and F, immunofluorescence intensity of cytoplasmic p11 and A2 signals was quantified using ImageJ and presented as the percentage of the signal detected in untreated cells (defined as 100%). G, the percentage of the fluorescence intensity of surface A2 after plasmin treatment, as compared with untreated cells, was estimated as indicated for E and F. H, HUVECs grown in 48-well culture plates were treated with plasmin for the indicated time periods, washed, and then assayed for t-PA-dependent plasmin generation, expressed as relative fluorescence units/min2.