Technical Reference #1786

1786.

Live Cell Analysis of Aquaporin-4 M1/M23 Interactions and Regulated Orthogonal Array Assembly in Glial Cells Jonathan M. Crane; Jeffrey L. Bennett; A. S. Verkman, University of California San Francisco, Journal of Biological Chemistry, 284(1786), (2009)
Link To Paper

Materials & Methods:
DNA Constructs Cell Culture and Transfections—DNA constructs encoding rat AQP4 (M1 and M23 isoforms and mutants thereof) and human AQP1 each containing a 10-residue Myc epitope (NH2-EQKLISEEDL-COOH) in the second extracellular loop were previously described (35). M23 with a hemagglutinin (HA) epitope (NH2-YPYDVPDYA-COOH) in place of the Myc epitope was generated by PCR amplification using rat M23 as template. A C-terminal serine residue was included to introduce a cloning restriction site and to generate the epitope tags of equal length (Fig. 1A). Constructs encoding non-epitope-tagged AQP4-M1 and M23 from rat mouse and human were PCR-amplified using whole-brain mRNA as template. All PCR fragments were ligated into mammalian expression vector pcDNA3.1 and fully sequenced.

Microscopic Technique
Freeze-fracture electron microscopy

Cell Type(s)
Glial cells