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Fig. 6. ICAM1 induced VEC phosphorylation in wt and mutant VEC. (A) CHO-ICAM1 cells were transfected with wt pEGFP-N'-VEC or not, grown to confluence and then starved. Cells were then subjected to ICAM1 crosslinking and VEC-GFP immunoprecipitated and analyzed by immunoblotting for phosphorylated tyrosine and VEC. C, untransfected controls; PV, sample from cells pretreated with pervandate (100 µM). (B) As described for A, except that the CHO-ICAM1 cells were transfected with wt or Y to F mutants of VEC as indicated. ICAM1 crosslinking was 10 minutes. (C) The sequence of the cytoplasmic domain of mouse VEC (as shown in Fig. 3A) has been used to predict tryptic peptides. Amino acids in small letters in peptide 11 are from the linker sequence to EGFP (which is not shown). Five out of the eleven peptides (bold) contain many phosphorylatable serine and tyrosine residues in line with our observation that VEC is strongly phosphorylated on serine and less so on tyrosine (data not shown). Note, in contrast to the report by Wallez et al. (Wallez et al., 2006) we have assumed that trypsin digestion does not occur when a proline is found at the carboxylic side of lysine or arginine. (D) CHO-ICAM1 cells were transfected with pEGFP-N'-VEC as described above. Cells were labeled with 32P and then subjected to ICAM1 crosslinking or not. VEC-GFP was immunoprecipitated and processed for tryptic peptide mapping. Arrows denote the position of crosslinking-specific phosphopeptides. The three maps displayed in a single row were chromatographed in the same tank and Rf values were directly comparable. Enlarged sections of the phosphopeptide maps showing ICAM1 crosslinking specific phosphopeptides are shown in supplementary material Fig. S2.
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