Identification of the amino-acid region involved in the intercellular interaction between the Na,K-ATPase β1 subunits

Abstract

SummaryEpithelial junctions depend on intercellular interactions between b 1 subunits of the Na + /K + -ATPase molecules of neighboring cells. The interaction between dog and rat subunits is less effective than the interaction between two dog b 1 subunits, indicating the importance of species-specific regions for b 1 -b 1 binding. To identify these regions, the species-specific amino acid residues were mapped on a highresolution structure of the Na

Septin Dynamics Are Essential for Exocytosis

“…This vector, which was constructed by Maura Hamrick in Dr. Douglas Fambrough's laboratory, was a generous gift from Dr. Liora Shoshani. The single L28A amino acid substitution in the DPPIV portion of this chimera generated a cleavage site for the endoplasmic reticulum (ER) signal peptidase, resulting in secretion of the soluble protein sec-␤1 that corresponds to the extracellular domain of the Na,K-ATPase ␤ 1 subunit (37,38). To determine the rate of secretion, aliquots of the culture medium were collected at the indicated time periods, and the amount of accumulated sec-␤1 was determined by Western blot analysis.…”

“…To control for the number of secreting cells and for the level of sec-␤1 expression, the amount of sec-␤1 in the medium was compared with the total amount in cell lysates. The antibody used for Western blot analysis reacts with the extracellular domain of the dog, but not human, Na,K-ATPase ␤ 1 subunit (38), resulting in specific detection of sec-␤1 without cross-reaction with the endogenous Na,K-ATPase ␤ 1 subunit in HEK-293 cell lysates.…”

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell–cell trans-dimerization of Na,K-ATPase β1 subunits

“…Cell aggregation was assessed by a hanging drop assay that was performed as previously described (Qin et al, 2005;Tokhtaeva et al, 2012). Cell suspensions containing 2.5×10 4 cells in 40 μl of cell culture medium with or without 20 μg/ml anti-β 1 antibody (clone M17-P5-F11), anti-E-cadherin mouse monoclonal antibody (clone DECMA-1; EMD Millipore) and an IgG1K control antibody (EMD Millipore), were placed as drops on the lid of a 35-mm culture plate and processed as previously described .…”

Recruitment of septin cytoskeletal proteins by Botulinum toxin A protease determines its remarkable stability

“…Membrane extracts were clarified by centrifugation (100,000 g, 1 h) at 4˚C. GFP-linked LCA or LCE were immunoprecipitated from total cell lysates (1-2 mg protein) or membrane extracts (0.5-1 mg protein) by using GFP antibody as described previously (Tokhtaeva et al, 2012b). The adherent proteins were eluted from the beads by incubation in 35 ml of SDS-PAGE sample buffer (4% SDS, 0.05% Bromophenol Blue, 20% glycerol, 1% b-mercaptoethanol in 0.1 M Tris pH 6.8) for 5 min at 80˚C.…”

“…Immunoprecipitated proteins eluted from the beads, and proteins of cell lysates or membrane extracts were separated by SDS-PAGE, transferred onto a nitrocellulose membrane (BioRad, Hercules, CA, USA) and detected by western blot analysis as described previously (Tokhtaeva et al, 2012b). Immunoblots were quantified by densitometry using Zeiss LSM 510 software, version 3.2.…”