Figure 4
DETA/NO pretreatment decreases sGC subunit protein levels in bovine chromaffin cells. A.- Immunodetection of sGC in soluble extracts from bovine chromaffin cells. Twenty micrograms of protein and 5 μL of molecular weight markers (prestained precision protein standards from BIO RAD) were electrophoresed and transferred to PVDF membranes and then sGC was detected by using 1 μg/mL of anti-sGC antibody or the antibody plus 80 ng/mL peptide β1 or antibody plus 60 ng/mL peptide α1.) B.- Cells were incubated with 50 μmol/L DETA/NO for the times indicated (0, 2, 4, 8, 24, 48 and 72 hours). Cells were then lysed and equal amounts of proteins (30 μg) corresponding to the soluble fraction of cell extracts fractionated by SDS-PAGE and transferred to PVDF membranes. The two sGC subunits (α1 and β1) were immunodetected using a polyclonal antibody as described in Methods. a-Actin was immunodetected to check for consistency in loading and transfer.