Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins

Fractions prepared from dog pancreas were sonicated (10 pulses, 1 s each) and centrifuged for 5 min at 10,000 g. Protein concentration in the supernatants was estimated using a protein assay kit (Micro-BCA; Pierce Chemical Co., Rockford, IL) followed by Coomassie staining of separated fractions. Aliquots of the fractions were then photometrically assayed for alkaline phosphodiesterase (PDE) activity using 5'-monophosphate p-nitrophenyl ester (Sigma Chemical Co., St. Louis, MO) as a substrate. Transfected yeast cells were grown to midlog phase, the cultures were harvested, and the pallets were resuspended in SDS--containing buffer. Cells were lysed with vigorous vortexing using glass beads. After low speed centrifugation, the supernatants were mixed with SDS protein sample buffer, subjected to SDS-PAGE, and transferred to nitrocellulose membranes for immanoblot analysis with connexin-specific anti-paptide antibodies. Bound antibodies were detected by coupled chemiluminescence. Immunoblots were stripped, reprobed with other connexin specific antibodies, and stained again following the instructions of the manufacturer (Amersham Corp.).