Hank s medium

For analysis of T cell profiles, PBMCs cultured for 48 h were resuspended (5 × 10⁵ cells/mL) in Hank's medium (Sigma, St. Louis, MO, USA), washed three times (400 ×g, 4°C, 10 min), and incubated in Hank's medium supplemented with 10% inactivated human AB+ serum. Subsequently, the samples were labeled with corresponding surface antibodies for each T cell profile. The cells were washed to remove excess antibody and then permeabilized and fixed with the addition of 500 μL Cytoperm/Cytofix (BD Biosciences, San Jose, CA, USA). Next, the samples were labeled with corresponding intracellular antibodies for each T cell profile and washed again to remove excess antibodies. Finally, the cells were resuspended in 500 μL PBS containing 0.5% paraformaldehyde and stored at 4°C in the dark until flow cytometry analysis. The samples were labeled with antibodies (BD Biosciences, San Jose, CA, USA) for T cell profiles, including Th1 (IFN-γ-FITC, T-bet-PE, and CD4-PerCP), Th2 (IL-4-FITC, GATA-3-PE, and CD4-PerCP), Th17 (IL-17-FITC, RORγT-PE, and CD4-PerCP), and Treg (CD25-FITC, FoxP3-PE, and CD4-PerCP). A FACSCalibur cytometer (Becton-Dickinson, Mountain View, CA, USA) was used for the acquisition of events (100,000 events/tube) and the data were analyzed using the CellQuest program (Becton-Dickinson).

The following reagents were used in the study: Hank’s medium (SIGMA, USA), gentamicin (SIGMA, USA), penicillin (SIGMA, USA), streptomycin (SIGMA, USA), PBS (SIGMA, USA), TCM-199 HEPES (SIGMA, USA), foetal bovine serum (FBS) (GIBCO, Scotland), mineral oil (SIGMA, USA), FSH (SIGMA, USA), β-oestradiol (SIGMA, USA), sodium pyruvate (MERCK, France), BSA fraction V (SIGMA, USA), BSA fraction FAF (SIGMA, USA), penicillamine (SIGMA, USA), hypotaurine (SIGMA, USA), epinephrine (SIGMA, USA), High Pure RNA Isolation Kit (Roche Diagnostics), SYBR Green (Roche Applied Science), Hybond-ECL nitrocellulose membranes (Amersham Biosciences), Trypan blue (SIGMA) antibody against HSP70 (Abcam, ab6535), antibody against OVGP1 (Abcam, ab74544), rabbit anti-mouse secondary antibodies (Santa Cruz Biotechnology, sc-2005), goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology, sc-2004), ECL Plus (Amersham Life Sciences), Fluoromount (Sigma USA).

The following reagents were purchased from Sigma-Aldrich: Hank’s medium, DME medium (DMEM), 199 medium (M199), pancreatin, gelatin, Triton X-100, 5-bromo-2′-deoxiuridine, anti-β-tubulin and anti-Rcan1 antibodies, and Herpud1, Mission #1 (universal negative control) siRNAs and Xestospongin C. The fluorescent probe Fura-2 AM, paraformaldehyde, and Hoechst 33342 were obtained from Thermo Fisher Scientific. The proteasome inhibitor MG-132 was purchased from Merck Millipore. Tunicamycin and the anti-Herpud1 antibody were from Enzo Life Science. Anti-IP3R antibody was purchased from Abcam. Anti-polyubiquitin Lys48 from Cell Signaling Technology and anti-β-MHC from Vector Laboratories. Secondary rabbit and rat anti-IgG antibodies conjugated with peroxidase were from Calbiochem. ECL chemiluminescent reagent for Western blotting was purchased from Amersham BioSciences. All SDS-PAGE materials and PVDF membranes were from Bio-Rad Laboratories. The mounting medium for fluorescence was purchased from DAKO Corporation. All other organic and inorganic reagents, salts, acids, and solvents were purchased from Merck unless otherwise specified.