HOSCN modulation of the AP-1 and Egr-1 pathways. (A) EMSA analysis of AP-1. HUVECs were stimulated with 10 μg/mL LPS or 150 μM HOSCN for 1 hour in M199 medium with 10% FBS. Two microgram aliquots of nuclear extracts were incubated with a radiolabeled, TF promoter–derived proximal AP-1 binding site oligonucleotide and analyzed as described for Figure 4. The arrow shows the mobility of the shifted probe and brackets the mobility of supershifted probe. Lanes designated “consensus” show the effect of adding 10-fold excess unlabeled AP-1 probe; “mutant,” the effect of adding 10-fold excess unlabeled mutant AP-1 probe; and “c-Jun” and “c-Fos,” the effect of adding monoclonal antibodies to the designated proteins. (B-D) EMSA analysis of Egr-1 and demonstration of upstream ERK1/2 kinase activation by HOSCN. (B) HUVECs were stimulated with 150 μM HOSCN (left panel) or 50 ng/mL PMA (right panel) for 1 hour in M199 medium with 10% FBS and nuclear extracts prepared. Five-microgram nuclear protein aliquots were incubated with a radiolabeled Egr-1 binding oligonucleotide probe and analyzed as described for Figure 4. The arrow shows the mobility of Egr-1–probe complex. Lanes designated “consensus” show the effect of adding 10-fold excess unlabeled Egr-1 probe; “mutant,” the effect of adding 10-fold excess unlabeled mutant Egr-1; and “Egr-1 Ab,” the effect of adding a polyclonal antibody for Egr-1. (C) Western blots of the nuclear extracts shown in panel B probed with an antibody for Egr-1 with SP1 as a loading control. (D) HUVECs were exposed to buffer (left panel) or 150 μM HOSCN (right panel) in complete medium for the indicated times, whole cell lysates prepared, and Western blots probed with an antibody specific for the phospho forms of the p44 and p42 components of Erk1/2, here designated “phospo-p44/42” (top row). p44/42 (Erk1/2) was probed as a protein loading control (bottom row).