Cell culture, stable cell lines and transfections
Cell lines used included CV-1 (ATCC CCL-70), HEK293 (ATCC CRL-1573), 293T (HEK 293 cells expressing the SV40 T-antigen), Jurkat (ATCC TIB-152), and MDCK strain II (Dog normal kidney epithelial cells). Jurkat cells were grown in RPMI 1640 (Life Technologies) supplemented with 10% fetal bovine serum. All other cell lines were grown in DMEM/F12 (1:1) (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Cells were cultured at 37°C in a humidified CO2 incubator.
Transient transfections were performed using FuGene™ 6 Transfection Reagent (Boehringer Mannheim) according to the supplier's instructions. Cells were incubated at 37°C for 18–24 hours before analysis.
Stable MDCK strain II cell lines were made expressing wild-type and carboxy-terminally mutated human GFP-LPP proteins, wild-type full length Scrib-GFP, or Scrib-GFP lacking all four PDZ-domains. Transfection of MDCK cells was performed using Lipofectamine 2000 Reagent (Life Technologies) according to the manufacturer's instructions. Transfected cells were selected in medium containing 250 μg/ml G418 (Life Technologies), and resistant colonies were isolated 10–14 days later. Individual clones were screened for expression of the respective GFP fusion proteins by Western blotting using a rabbit polyclonal anti-GFP antibody (Tebu Bio).