Anti ido

Flow cytometry: commercial antibodies anti-CD86-FITC (clone 2231 FUN-1), CD274 (PD-L1)-FITC (clone MIH1), CD273 (PD-L2)-PE (clone MIH-18), HLA-DR-PE-Cy7 (clone L243), IFN-γ-FITC (clone 4SB3) were purchased from BD Biosciences; CD83-PerCP-Cy5.5 (clone HB15a) was purchased from Beckman Coulter; CD80-FITC (clone MAB104), CD40-PerCP-eFluor710 (clone 5C3), CD1a-PE-Cy7 (clone HI149) and CD4-PE-Cy7 (clone RPA-T4) were purchased from eBioscience; TLR2-FITC (clone T2.5), TIM-3-PE (clone F38-2E2), IL-10-PE (clone JES3-9D7), KI-67-PE (clone Ki-67) were purchased from BioLegend; CD14-PE-DL594 (clone MEM-15), CD11c-APC (clone BU15), CD3-AF700 (clone MEM-57), CD8-PE-Dy590 (clone MEM-31) were purchased from Exbio; CD85k (ILT-3)-PE (clone 293623), CD85d (IL-T4)-FITC (clone 287219) were purchased from R&D Systems. For western blot, anti-p-p38 MAPK, anti-p-ERK1/2, anti-p-JNK/SAPK, anti-p-IκB-α, anti-IDO, anti-p-mTOR, anti-p-STAT3, anti-p-p70S6K, anti-p38 MAPK, anti-ERK1/2, anti-JNK/SAPK and anti-STAT3 Ab were purchased from Cell Signaling Technology; anti-actin was from BioLegend.

After imDCs (3 × 10⁶/mL) cultured with or without LPS were stimulated with 20% DCHJ for 48 h, cells were washed twice with PBS and lysed with lysis buffer (Solarbio, Beijing, China). Proteins in the cell lysates were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membranes (Solarbio, Beijing, China). The membranes were then blocked with 5% nonfat milk in Tris-Tween-20 (TBST) for 2.5 h at room temperature and incubated with anti-IDO (1 : 1000 dilution, Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin (1 : 100000 dilution, Cell Signaling Technology, Beverly, MA, USA) antibodies at 4°C overnight. After three washes, membranes were then incubated with an HRP-conjugated secondary antibody (1 : 2500 dilution) for 2 h at room temperature. The immunoreactive protein bands were visualized using an enhanced chemiluminescence detection kit (Millipore, Billerica, MA, USA).