Separation of regulatory myosin light-chain isoforms by 2-D gel electrophoresis.
After ECs treated with buffer or TNF-α for 24 hours were washed twice, neutrophils (neutrophil to EC ratio, 1:1) were added to TNF-α–treated ECs and allowed to adhere for 2, 6, or 15 minutes. After two washes in cold PBS, the cells were lysed and the phosphorylation myosin light-chain isoforms was analyzed using 2-D gel electrophoresis as described in “Materials and methods.” Only the region of the gels containing regulatory myosin light-chain isoforms is shown. (A-F) Myosin light-chain isoforms in untreated ECs (A). ECs treated with 1 U/mL thrombin for 30 minutes (B). Twenty-four-hour TNF-α–treated ECs (C) or 24-hour TNF-α–treated ECs with neutrophils adherent for 2, 10, or 15 minutes (D-F). (G) Quantification of mol PO4/mol myosin light-chain molecules; U, unphosphorylated regulatory myosin light chain; M, monophosphorylated regulatory myosin light chain; D, diphosphorylated myosin light chain.
