[23] Expression and properties of Rab25 in polarized Madin-Darby canine kidney cells

This chapter discusses the expression and properties of Rab25 in polarized Madin-Darby canine kidney cells. In Madin-Darby canine kidney (MDCK) cells, Rab25 levels are very low, whereas endogenous Rablla levels are relatively high. Therefore, to study Rab25 in MDCK cells, cell lines are isolated stably transfected with the wild-type sequence of rabbit Rab25. To detect Rab25, monoclonal antibodies are developed specific for Rab25 and nonreactive against Rab11a. A double-screening enzyme-linked immunosorbent assay (ELISA) protocolis developed to isolate monoclonal antibodies against Rab25 that do not react with Rablla. To visualize the three-dimensional distribution of Rab proteins in polarized cells, the stably transfected Rab25- expressing 2A3 cell line is grown to confluence on Transwell clear filters and maintained for three days following confluence. Out of 20 lines screened, six lines were found to contain a transfected Rab25 message. The 2A3 line utilized for characterization of the association of Rablla and Rab259 was one of the two lowest expressing cell lines. To compare the effects of stable overexpression of Rab25 on endogenous message levels, Rab25 and Rablla mRNA levels were compared between mock pCB6-transfected cells and the Rab25-transfected 2A3 line.