Stat5

Cells were then lysed at the indicated time points in a lysis buffer containing protease inhibitors, and the resultant cell lysates were separated by SDS-PAGE under reducing conditions and transferred to polyvinylidene difluoride membrane (Merck Millipore). The membrane was blocked, probed with antibodies against p19 (R&D Systems), EBI3 (Santa Cruz), CD5L (D-11 from Santa Cruz, GeneTex), STAT1 (Transduction Laboratories), STAT3 (Santa Cruz), STAT5 (Santa Cruz), AKT (Cell Signaling), ERK (Cell Signaling), pY-STAT1 (Cell Signaling), pY-STAT3 (Cell Signaling), pY-STAT5 (Cell Signaling), pAKT (Cell Signaling), pERK (Cell Signaling), or actin (Sigma-Aldrich), followed by an appropriate secondary antibody conjugated to horseradish peroxidase and visualized with an enhanced chemiluminescence detection system (GE Healthcare) per the manufacturer’s instructions. Immunoreactive bands were detected with a ChemiDoc XRS (Bio-Rad). To detect phosphorylation, naive CD4⁺ T cells (5 × 10⁵ cells/ml) were stimulated with plate-bound anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) under Th conditions for 3 days. The resultant cells were washed, rested in 10% FBS medium for 6 h and stimulated with various molecules for the indicated times, followed by western blotting with anti-pY-STATs, anti-pAKT and anti-pERK, and subsequently with antibodies against their total molecules.

Cells were lysed in SDS Lysis buffer (12 mM Tris-Cl, pH 6.8, 5% glycerol, and 0.4% SDS), and the protein concentrations were measured with the SMART BCA Protein Assay kit (iNtRON Biotechnology, Korea). The proteins were resolved with SDS-polyacrylamide gel electrophoresis followed by transfer to PVDF membranes (Millipore, Billerica, MA, USA) and incubated overnight with the appropriate antibodies. The antibodies used were as follows: phospho-STAT5 (p-STAT5; rabbit polyclonal IgG; #9351, 1:1,000), cyclin D1 (rabbit polyclonal IgG; #2922, 1:1,000), and PARP (rabbit polyclonal IgG; #9542, 1:1,000) from Cell Signaling Technology (Danver, MA, USA), STAT5 (rabbit polyclonal IgG; #sc-835, 1:1,000) from Santa Cruz (Santa Cruz, CA, USA), and β-actin (mouse monoclonal IgG; # A5441, 1:5000) from Sigma-Aldrich. Goat anti-rabbit IgG (#111-035-003; 1;5,000) and anti-mouse IgG (#115-035-033; 1:5,000) secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

An equal number of cells were collected and subjected to immunoblot analysis to determine protein expression levels of Bcl-6 (BD Biosciences, 561520, dilution 1:500), Blimp-1 (Genscript, A01647, dilution 1:500), STAT5 (Santa Cruz, sc-835, dilution 1:5,000), phospho-STAT5 (pY694, BD Biosciences, 611964, dilution 1:5,000) and V5-tagged proteins (Invitrogen, R960-25, dilution 1:5,000). In brief, separation of lysates by SDS–polyacrylamide gel electrophoresis was followed by immunoblot analysis. GAPDH (Santa Cruz, sc-25778, dilution 1:2,500) or β-actin (Genscript, A00730, dilution 1:10,000) expression was monitored to ensure equal protein loading. Additional information for antibodies can be found in Supplementary Table 3 and uncropped versions of all immunoblots are provided in Supplementary Fig. 5.