HeLa, HEK293T, and COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen, San Diego, CA) supplemented with 10% fetal bovine serum (Invitrogen) Selleck DMXAA containing penicillin and streptomycin. The Tac backbone construct was kindly provided by Dr. John Marshall

(Brown University, Providence, RI). The schematic structure of the TacCterm chimeras, consisting of the extracellular and transmembrane domains of Tac and the C-terminal tail of BSEP, is shown in Fig. 1. The Tac coding sequence was amplified by polymerase chain reaction (PCR) insertion of EcoRV and XbaI sites for subcloning into pcDNA3 (Invitrogen). TacCterm chimeras were constructed by a two-stage PCR method, using two sets of overlapping primers and ligating into the Tac construct using EcoRV and the XbaI site. The C-terminal tail of BSEP encoding residues D1284 to S1321 was amplified using human BSEP (kindly provided by Selleck LY2157299 Dr. Bruno Stieger, University Hospital, Zurich, Switzerland). Deletion mutants of TacCterm (del 1298-1316; del1308-1316) and alanine substitutions in the following mutants were generated by site-directed mutagenesis: YY (Y1310A, Y1311A); LM (L1303A, M1304A); and

LMYY (L1303A, M1303A, Y1310A, Y1311A). A shorter version of the C-terminal BSEP, Tac8A-YY, was also generated by inserting eight alanines and the corresponding two residues G1308AYYKLV1314). All constructs were confirmed by DNA sequencing. Human full-length BSEP was amplified by PCR and inserted into the EcoRV site of the pWAY21-EGFP expression vector provided by Dr. Anton Bennett (Yale University, New Haven, CT). Mutant GFP-BSEP (Y1310A/Y1311A) was 上海皓元 generated by site-directed mutagenesis using the QuickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). HEK293T cells were plated on poly-L-lysine coverslips and transiently transfected using LipofectAMINE 2000 reagent (Invitrogen) for 18 hours. Cells were washed with ice-cold phosphate-buffered saline (PBS; with Mg and Ca) and labeled with mouse anti-Tac antibody (IL-2Rα, 0.5 μg/mL, 30 minutes, 4°C; BD Transduction Laboratories, San Jose, CA) in labeling buffer (PBS/Mg/Ca/0.2% bovine serum albumin [BSA]).

Internalization was initiated by warming to 37°C, carried out for the indicated time, and then stopped by washing repeatedly with ice-cold labeling buffer. Cells were fixed in 4% paraformaldehyde, washed with PBS, and permeabilized with 0.1% Triton X-100. TacCterm–anti-Tac complexes were detected with Alexa 488 or Alexa 568 anti-mouse secondary antibody (1:500; 1 hour), and fluorescent images were acquired on an LSM 510 confocal microscope (Carl Zeiss Inc., Thornwood, NY). Internalization of Tac chimeras was examined after cotransfection with the dominant-negative construct K44A dynamin (provided by Dr. Pietro de Camilli, Yale University) and with wild-type Rab5a-DsRed and dominant-negative N133I Rab5a-DsRed, kindly provided by Dr. Richard Pagano (plasmids 13050, 13051, Addgene, Cambridge, MA).