2.1. Cell lines and reagents
Human embryonic kidney cells (HEK293T: CRL-11268, ATCC) were cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and penicillin (100 U/ml)/ streptomycin (100 μg/ml, Gibco). Leptomycin B (LMB) was obtained from LC Laboratories.
Cloning of human GFP-NLRC5, GFP-CIITA and the NLRC5 import mutants NLS I (RRK133/134/135A) and NLS II (KR121/122A) has been described previously [17
]. Combining NLSI and NLSII resulted in the double mutant DM (KR121/122A , RRK132/133/134A). All point mutations were introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Cloning of the NLRC5 Walker A, B and AB constructs has also been described previously [17
]. To rescue the nuclear import defect of the Walker A mutant, SV40 NLS (PKKKRKV) sequences were appended at both ends of NLRC5 by PCR using the following primers: F, 5’-ATATGAATTCGGAGCTCCAAAGAAGAAGCGTAAGGTAGACCCCGTTGGCCTCCAGC TCGG-3’; R, 5’-ATATTCTAGATTATACCTTACGCTTCTTCTTTGGAGCAGTACCCCAAGGGGCCTG-3’ (Walker A2xNLS). Using the same primers, a NLRC5 WT construct containing two SV40 NLSs was constructed and used as a control (WT2xNLS). The MHC class I and class II reporter gene constructs used in reporter gene assays were a kind gift from Dr. P.J. van den Elsen (Leiden University).
2.3.Transfections and generation of stable cell lines
HEK293T cells were transiently transfected using polyethylenimine (1 mg PEI/ ml, pH 7.2, Polysciences, Inc.) at a ratio of 1:3 (DNA:PEI). Medium was changed the following day and cells were analyzed 48 hrs post transfection. To select for stable integration of expression plasmids, 2 mg/ml G418 (Gibco) was added to the culture medium 24 hrs after transfection for 10 days. GFP-positive cells were further enriched by cell sorting using a MoFlo high-speed sorter (Dako).
HEK293T cells were grown overnight on glass coverslips coated with poly-L-lysine (Sigma-Aldrich). Prior to imaging cells were rinsed with PBS and stained with Hoechst 33342 (1 μg/ml, Invitrogen) to stain the nuclei before fixing with 10% phosphate buffered formalin. Coverslips were mounted onto glass slides using ProLong Gold Antifade Reagent (Invitrogen). Epifluorescence microscopy was performed using a Nikon Eclipse E800 (Nikon Instruments). For confocal microscopy, cells were cultured in a coverslip-mounted 20 mm dish (MatTek) 24 hours prior to imaging. Cells were stained with Hoechst 33342 for 10 min and then washed with PBS just prior to imaging. Images were acquired with the Eclipse Ti spinning disk confocal microscope (Nikon) using the 100X objective (1.40 NA) and the ORCA-ER camera (Hamamatsu). GFP was imaged using the solid phase argon laser at 488nm and a 520/30 band pass filter. The Hoechst 33342 stained nuclei were imaged using the solid phase UV laser at 405 nm and a 475/30 band pass filter. Image analysis was performed using ImageJ (NIH).
2.5. Luciferase assay
HEK293T cells were split into 24-wells and co-transfected with 300 ng of either GFP, GFP-NLRC5, or the corresponding mutant expression plasmids and 100 ng of the indicated luciferase reporter construct. 50 ng per well of Renilla plasmid (pRL-null, Promega) was included to allow for normalization of transfection efficiency. Cells were harvested 48 hrs post transfection, and cell lysates were analyzed using the dual-luciferase system (Promega), according to the manufacturer’s instructions.
2.6. Quantitative real-time PCR analysis
RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg RNA using the qScript Flex cDNA synthesis kit (Quanta Biosciences), and RNA expression was quantified on the 7300 Real-Time PCR System (Applied Biosystems) using the PerfeCTa SYBR Green SuperMix with ROX (Quanta Biosciences), and analyzed using the 7300 System SDS Software (Applied Biosystems). The primers used for amplification have been described previously [17
2.7. Western blot analysis
Whole cell extracts were prepared using 1x Cell Lysis Buffer (Cell Signaling) supplemented with 1 mM DTT, and 1 mM PMSF. Following SDS-polyacrylamide gel electrophoresis using 4–12% gradient gels (Invitrogen), proteins were transferred to nitrocellulose membranes (HyBond ECL, Amersham) for 2 hrs at 80V. The following antibodies were used for protein detection: anti-MHC class I heavy chain (3B10.7, a kind gift from Dr. P. Cresswell, Yale University), anti-GFP (JL-8 Clontech), anti-β-actin (I-19, Santa Cruz). Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific), and imaged using the Molecular Imager ChemiDoc XRS+ System (Bio-Rad) or exposed to autoradiography film (Denville). Quantification was performed using ImageQuant (Molecular Dynamics).
2.8. Flow cytometry
To determine HLA class I surface expression, cells were stained with phycoerythrin (PE)-conjugated anti-HLA-A,B,C (W6/32, BioLegend) following fixation with 1% paraformaldehyde. FACS analysis was performed using the FACSCalibur system (BD Biosciences) and analyzed using the FlowJo software (Tree Star).
2.9. Statistical analysis
Data were subjected to Student's t test for analysis of statistical significance and plotted using Prism (GraphPad). Results are given as the mean ± SD. A P-value of < 0.05 was considered to be significant.