PDLIM2−/− mice were backcrossed with BALB/c mice at least 10 generations for pure BALB/c background. PDLIM2−/− BALB/c mice and control PDLIM2+/+ BALB/c mice were housed under specific pathogen-free conditions at the Hillman Cancer Center of the University of Pittsburgh Cancer Institute. Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pittsburgh.
Experimental autoimmune encephalitis (EAE) induction and clinical scoring
Six to eight-week-old female mice were immunized subcutaneously with PLP180–199 peptide (200 μg/mouse, Genemed Synthesis Inc.) emulsified in CFA containing Mycobacterium tuberculosis H37Ra (500 μg/mouse, BD Diagnostics). Mice also received 300 ng of pertussis toxin (List Biological Laboratories) intraperitoneally (i.p.) at the time of immunization and 48 hours later. Mice were monitored daily for clinical signs of paralysis and scored as follows: 0, no clinical signs; 1, limp tail; 2, weak/partially paralyzed hind legs; 3, limp tail and complete paralysis of hind legs; 4, complete hind and partial front leg paralysis; 5, complete paralysis or moribund state.
Adoptive transfer of CD4+ T cells for induction of EAE
Lymph nodes and spleens were harvested from PDLIM2+/+ or PDLIM2−/− mice immunized with PLP180–199, and lymph node cells and splenocytes were cultured in vitro with 1 μM PLP180–199 and IL-2 for 72 h. CD4+ T cells were then positively selected by MACS separation using magnetic CD4+ microbeads (Miltenyi Biotec, Auburn, CA) per manufacturer’s instructions. 5 x 106 CD4+ T cells were adoptively transferred by intravenous (i.v.) injection into SCID recipients on day 0. On day 2, mice received an i.p. injection of pertussis toxin (250 ng), and mice were then monitored for symptoms of disease.
CD4+ th cell purification and in vitro differentiation
Naive CD4+CD25- T cells were first isolated from splenocytes using CD4+ T-cell Isolation Kit (Miltenyi Biotec.) and then sorted out by FACSAria (BD Biosciences). Purified naive CD4+CD25- T cells were stimulated with plate-bound anti-CD3 and anti-CD28 (1 μg/ml) under Th1 (mIL-2 10 ng/ml, mIL-12 10 ng/ml), Th2 (IL-4 10 ng/ml, anti-IFNγ 10 μg/ml), Th17 (anti-IFNγ 10 μg/ml, anti-IL-4 10 μg/ml, hIL-6 10 ng/ml, hTGF-β 10 ng/ml) or Treg (hTGFβ, 10 ng/ml, anti-IL-4 10 μg/ml, anti-IFNγ 10 μg/ml) (BD Biosciences or eBioscience) polarizing condition. 72 hours after the initial stimulation, the cells were subjected to intracellular cytokine staining (ICS)/flow cytometry analysis and quantitative real-time RT-PCR (QRT-PCR) as described below.
ICS and flow cytometry
T cells were stimulated for 5 hours with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of intracellular transport inhibitor monesin (10 μg/ml; Sigma), followed by fixation with paraformaldehyde (2%) and permeablization with saponin (0.5%). Cells were then treated with anti-IFN-γ-FITC (XMG1.2), anti-IL-4-PE (11B11), anti-IL-17-PE (TC11-18 H10), and anti-Foxp3–FITC (FJK-16 s) (BD Biosciences or eBioscience). Data were acquired using FACSCalibur (BD Biosciences) and analyzed using CellQuest software (Becton Dickinson) as described previously .
Total RNA was prepared with TRIZOL reagent and cDNA was generated with SuperScript II reverse transcriptase (Invitrogen), followed by real-time PCR assays using Fast start SYBR Green reagents (Roche) as described [24, 25]. The gene-specific primer pairs were: IFN-γ, 5’-TTCTTCAGCAACAGCAAGGCGAA-3’ and 5’-TGAATGCTTGGCGCTGGACCTG-3’; TNF-α, 5’-GATGAGAAGTTCCCAAATGGC-3’ and 5’-ACTTGGTGGTTTGCTACGACG-3’; TGF-β, 5’-TGACGTCACTGGAGTTGTACGG-3’ and 5’-GGTTCATGTCATGGATGGTGC-3’; IL-4, 5’-AGGGACGCCATGCACGGAGAT-3’ and 5’-GCGAAGCACCTTGGAAGCCCTAC-3’; IL-5, 5’-AGCACAGTGGTGAAAGAGACCTT-3’ and 5’-TCCAATGCATAGCTGGTGATTT-3’; IL-10, 5’-AGCTGAAGACCCTCAGGATGCG-3’ and 5’- TCATTCATGGCCTTGTAGACACCTTG-3’; IL-13, 5’-GGCTCTTGCTTGCCTTGGTG-3’ and 5’-TCCATACCATGCTGCCGTTG-3’; IL-17, 5’-CTCAGACTACCTCAACCGTTC-3’ and 5’-TGAGCTTCCCAGATCACAGAG-3’; IL-21, 5’-ATCCTGAACTTCTATCAGCTCCAC-3’ and 5’-GCATTTAGCTATGTGCTTCTGTTTC-3’; IL-22, 5’-TCCGAGGAGTCAGTGCTAAA-3’ and 5’-AGAACGTCTTCCAGGGTGAA-3’; β-actin, 5′-ACCCGCGAGCACAGCTTCTTTG-3’ and 5’-CTTTGCACATGCCGGAGCCGTTG-3’. Expression levels of each gene were normalized to that of β-actin.
Enzyme-linked immunosorbent assay (ELISA)
Cell nuclear fractions were prepared and added to 96-well plate precoated with anti-RelA, anti-STAT3 or anti-STAT4. After overnight incubation at 4 °C, plates were washed extensively with PBS containing 0.1% Tween 20 (PBST), and horseradish peroxidase-conjugated secondary antibodies were added and incubated for 1 hour at room temperature. After extensive wash with PBST, a colorimetric substrate 2’2-azinobis(3-ethylenzthiazoline-6-sulfonic acid) (ABTS) was added and incubated for 15 minutes. The reaction was stopped by addition of 100 μL 1% sodium dodecyl sulfate (SDS). The optical density at 405 nm (OD405) was measured with an automated plate spectrophotometer (Thermo Lab Systems).
Immunoblotting (IB) and immunoprecipitation (IP) assays
Nuclear extracts were subjected to SDS-PAGE and IB, or IP using the indicated antibodies before SDS-PAGE and IB as described before [26, 27].
In vivo ubiquitin conjugation assay
Cytoplasmic and nuclear extracts were prepared from HTLV-I-transformed T cells or 293 cells transfected with HA-STAT3 together with Flag-tagged ubiquitin in the presence or absence of Myc-PDLIM2, immediately followed by IP using anti-HA. The ubiquitin-conjugated STAT3 pulled down by IP was detected by IB using anti-Flag .
Protein stability assay
Cells were treated with 10 μM CHX, followed by chase of the indicated time period in the presence or absence of MG132, and IB to detect the indicated proteins .
Data were reported as mean ± standard deviation (SD). The Student’s t test (two tailed) was used to assess significance of differences between two groups, and p values ≤ 0.05 and 0.01 were considered statistically significant and highly statistically significant, respectively.