P ser235.236 s6 ribosomal protein d57 2 2e

After blocking Fc receptors with anti-CD16/CD32 Ab (BD Bioscience), surface staining of isolated single cells was performed in PBS with 2% FBS with the following monoclonal antibodies (mAb) or reagent: PE anti-CD138 (282-2, BioLegend; 1:100 dilution), APC-Cy7 anti-B220 (RA3-6B2, BioLegend; 2:100), 7-AAD (BioLegend; 3:100), FITC anti-GL-7 (GL7, BD Bioscience; 0.5:100), PE anti-PD-1 (J43, BD Bioscience; 1.5:100), APC anti-Fas (15A7, eBioscience), biotinylated mAb for CD8a (53-6.7, BioLegend), CD11c (N418, BioLegend), CD19 (1D3, eBioscience), and CXCR5 (2G8, BD Bioscience; 5:100), and streptavidin (SA)-APC (BioLegend; 1.5:100). For phosphorylated-protein detection, cells were stained with p-Ser235.236-S6 ribosomal protein (D57.2.2E, Cell Signaling; 1:100) and p-Thr37/46-4E-BP1 (236B4, Cell Signaling; 1:100) after preparing with BD Phosflow lyse/fix buffer and perm buffer III (BD Bioscience), according to the manufacturer’s protocol. For MitoTracker staining, cultured cells stained with 30 nM MitoTracker Green (Invitrogen) and 30 nM MitoTracker DeepRed (Invitrogen) were incubated in a CO₂ incubator at 37°C for 30 min, according to the manufacturer’s protocol. Stained cells were analyzed and sorted using a FACSVantage SE (Becton-Dickinson, Franklin Lakes, NJ, USA). Data were further assessed with FlowJo (Tree Star, Ashland, OR, USA).

For staining of phosphorylated proteins and metabolic enzymes or transcription factors, cells were fixed with Cytofix Fixation buffer (BD Bioscience, Franklin Lakes, New Jersey) for 10 min at 37 °C, 5 % CO₂. T cells were permeabilized with Phosflow Perm Buffer III (BD Bioscience, Franklin Lakes, New Jersey) for 30 min on ice, before cells were stained with antibodies against surface markers and phospho-specific antibodies [p_Ser727STAT1 (A15158B) (Biolegend, San Diego, California), p_Tyr705STAT3 (4/P-STAT3) (BD Bioscience, Franklin Lakes, New Jersey), p_Tyr694STAT5 (47/Stat5) (BD Bioscience, Franklin Lakes, New Jersey), p_Ser2448mTOR (MRRBY) (Thermo Fisher Scientific, Waltham, Massachusetts), p_Ser235/236S6 Ribosomal Protein (D57.2.2E) (Cell Signaling Technology, Danvers, Massachusetts), LDHA (C4B5) (Cell Signaling Technology, Danvers, Massachusetts) or SDHA (D6J9M) (Cell Signaling Technology, Danvers, Massachusetts)].