Differential Regulation of TauT by Calcitriol and Retinoic Acid via VDR/RXR in LLC-PK1 and MCF-7 Cells - Advances in Experimental Medicine and Biology - page 274

27.2 Materials and Methods

27.2.1 Cell Lines and Cell Culture

HK293, MDCK, LLC-PK1 renal cells, and MCF-7 human breast cancer cells were
obtained from the American Type Culture Collection. The cells were grown in
Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal
bovine serum (Gibco), 50 units/ml penicillin, and 50 mg/ml streptomycin. All cells
were maintained in a humidified 37°C incubator with 5% CO 2, fed every 3 days with
a complete medium, and subcultured when confluence was reached.

27.2.2 Measurement of Taurine Transport

Taurine transport studies were performed on confluent monolayers 3 days after
seeding cells. The cells were rinsed with Earle's balanced salt solution (EBSS) at
37°C. Uptake was initiated by the addition of an uptake buffer (2 mM KCl, 1 mM
MgCl 2 , 96 mM NaCl, 1.8 mM CaCl 2 , 5 mM Hepes, pH 7.6) to which 50 m M unla-
belled taurine and 0.5 m Ci/ml 14 C-taurine (Perkin Elmer, Boston, MA) were added.
After a 30-min period of incubation at room temperature, uptake was terminated by
the removal of uptake buffer followed by three rapid washes with cold EBSS. Cells
were dissolved in 1% SDS in 0.2 N NaOH and radioactivity was counted in a
Packard 2000-CA Liquid Scintillation Analyzer.

27.2.3 Western Blot Analysis

Cells were lysed in 50 ml M-PER mammalian protein extraction reagent (Pierce,
Inc., Rockford, IL) supplemented with a protease inhibitor cocktail for use with
mammalian cell and tissue extracts (Sigma). The lysates were cleared by centrifuga-
tion at 14,000 × g for 2 min, and the supernatants were transferred to clean tubes.
Equal amounts of protein (50 mg) were separated by electrophoresis on a 12% SDS-
polyacrylamide gel and transferred to a nitrocellulose membrane (Millipore,
Bedford, MA) using a semi-dry electrophoretic transfer system (Bio-Rad).
Membranes were incubated in 5% nonfat dry milk in Tris base/sodium chloride
(TBS) buffer with 0.2% Tween 20 (TBST) at 4°C overnight. The membranes were
incubated with primary antibodies for 1 h at room temperature. Blots were washed
with TBST and incubated with horseradish peroxidase-linked secondary antibody
(Sigma) for another hour, and then proteins of interest were detected using a chemi-
luminescence detection kit (Pierce, Inc.).